USE OF CIS-EPOXYEICOSATRIENOIC ACIDS AND INHIBITORS OF SOLUBLE EPOXIDE HYDROLASE TO TREAT CONDITIONS MEDIATED BY PBR, CB2, and NK2 RECEPTORS

ABSTRACT

The invention relates to the discovery that cis-epoxyeicosatraenoic acids (EETs) bind to and act as agonists of peripheral benzodiazepine receptor and the cannabinoid CB 2  receptor. The invention provides methods of reducing symptoms of conditions whose activity is mediated by these receptors, including inhibiting anxiety, inhibiting the growth of cancer cells expressing peripheral benzodiazepine receptors, and reducing oxygen radical damage to cells, by contacting the cells with a cis-epoxyeicosantrienoic acid, an inhibitor of soluble epoxide hydrolase (sEH), or both. The invention further provides methods of inhibiting irritable bowel syndrome by administering to individuals with inhibiting irritable bowel syndrome a cis-epoxyeicosantrienoic acid, an inhibitor of soluble epoxide hydrolase (sEH), or both. In some embodiments, the method comprises administering to the individual a nucleic acid which inhibits expression of sEH.

CROSS-REFERENCES TO RELATED APPLICATIONS

NOT APPLICABLE.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This application claims priority from and benefit of U.S. ProvisionalApplication Ser. No. ______, Attorney Docket No. 02307O-168800US, filedDec. 15, 2006, the contents of which are incorporated herein byreference.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

It would be useful to have additional methods of decreasing anxiety,inhibiting the proliferation of cancer cells, of reducing irritablebowel syndrome, and of increasing endogenous neurosteroid production.

The present invention fills these and other needs.

BRIEF SUMMARY OF THE INVENTION

In a first group of embodiments, the invention provide methods ofrelieving symptoms of a condition selected from the group consisting ofanxiety, panic attack, agitation, status epilepticus, other forms ofepilepsy, alcohol or opiate withdrawal, insomnia, or mania in a subjectin need thereof, by administering to the subject an effective amount ofan agent or agents selected from the group consisting of acis-epoxyeicosantrienoic acid (“EET”), an inhibitor of soluble epoxidehydrolase (“sEH”), and a combination of an EET and an inhibitor of sEH,thereby relieving the symptoms of the condition in the subject. In someembodiments, the agent is an EET. In some embodiments, the EET isselected from the group consisting of 14,15-BET, 8,9-EET, 11,12-EET or5,6-EET. In some embodiments, the agent is an inhibitor of sEH. In someembodiments, the condition is anxiety.

In a further group of embodiments, the invention provides methods ofinhibiting growth of cancer cells expressing peripheral benzodiazepinereceptors (PBR) or CB₂ receptors. The methods comprise contacting saidcells with an effective amount of an agent or agents selected from thegroup consisting of a cis-epoxyeicosantrienoic acid (“EET”), aninhibitor of soluble epoxide hydrolase (“sEH”), and a combination of anEET and an inhibitor of sEH, thereby inhibiting the growth of the cancercells. In some embodiments, the cancer cells are glioma cells. In someembodiments, the cells are astrocytoma cells. In some embodiments, thecells are breast cancer cells. In some embodiments, the agent is an EET.In some embodiments, the EET is selected from the group consisting of14,15-EET, and 11,12-BET. In some embodiments, the agent is an inhibitorof sEH. In some embodiments, the EET or the inhibitor of sEH, or both,are contained in a material which releases the EET or the inhibitor, orboth, over time.

In yet a further group of embodiments, the invention provides methods ofreducing oxygen radical damage to cells. The methods comprise contactingsaid cells with an effective amount of an agent or agents selected fromthe group consisting of a cis-epoxyeicosantrienoic acid (“EET”), aninhibitor of soluble epoxide hydrolase (“sEH”), and a combination of anEET and an inhibitor of sEH, thereby reducing oxygen radical damage tothe cells. In some embodiments, the agent is an EET. In someembodiments, the EET is selected from the group consisting of 14,15-BET,8,9-EET and 11,12-EET. In some embodiments, the agent is an inhibitor ofsEH. In some embodiments, the EET, or said inhibitor of sEH, or both,are administered by applying to the skin a topical formulationcomprising the EET or the inhibitor of sEH, or both. In someembodiments, the topical formulation further comprises a sunscreen orsunblock.

In still a further group of embodiments, the invention provides methodsof relieving symptoms of irritable bowel syndrome (IBS) in a subject inneed thereof. The method comprises administering to the subject aneffective amount of an agent or agents selected from the groupconsisting of a cis-epoxyeicosantrienoic acid (“EET”), an inhibitor ofsoluble epoxide hydrolase (“sEH”), and a combination of an EET and aninhibitor of sEH, thereby relieving symptoms of IBS in the subject. Insome embodiments, the agent is an EET. In some embodiments, the EET isselected from the group consisting of 14,15-EET, 8,9-EET, and 11,12-EET.In some embodiments, the agent is an inhibitor of sEH.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 is a graph showing the results of in vitro assays showingthat EETs displace the high affinity PBR ligand [H³] PK 11195 in a dosedependent manner. X axis: Micromolar concentration of EETs. Y axis: %Inhibition of binding of [H³] PK 11195. Filled diamonds: 5,6-EET. Filledcircles: 11,12-EET. Filled triangles: 14,15-EET. Filled squares: Mixtureof epoxyeicosatrienoic acid methylesters (“EETs-me”).

FIGS. 2A-2D. FIGS. 2A-2D show the results of in vivo assays showing thatsEHI elicited analgesia in rats can be blocked by steroid synthesisinhibitors. FIG. 2A shows the effect on animals administered the steroidsynthesis inhibitor aminogluthetimide (“AGL”), while FIG. 2B shows theeffect of animals administered the steroid synthesis inhibitorfinasteride (“FIN”). FIG. 2C shows that show that AGL and FIN bythemselves have no impact on the baseline response shown by controlanimals, which FIG. 2D shows that they do not they modify the responseof animals to LPS administration. For each of FIGS. 2A-D, the Y axisshows hindpaw thermal withdrawal latencies (“TWL”) of the animals in thestudy reported in the Figure as a percentage of TWL prior to anytreatment (“Baseline”). The X axis shows the various points in time atwhich measurements of TWL were taken. “BL” means starting (“base line”)measurement taken before administration of agents to the animals. FIG.2A: Hollow diamonds: animals treated only with lipopolysaccharide(“LPS”) (n=16). “X” with vertical line: animals treated with LPS+aninhibitor of sEH called “AEPU” (n=6). “X”: animals treated with LPS andAGL (n=6). “+” sign: animals treated with AGL, LPS, and AEPU (n=7). FIG.2B: Hollow diamonds: animals treated only with LPS (n=16) (same data asin FIG. 2A). “X” with vertical line: animals treated with LPS+aninhibitor of sEH called “AEPU” (n=6) (same data as in FIG. 2A). “X”:animals treated with LPS and FIN (n=6). “+” sign: animals treated withFIN, LPS, and AEPU (n=8). FIG. 2C: Filled diamonds: control (untreated)animals (n=6). Filled squares: animals treated with AEPU, but not LPS(n=6). Filled triangles: animals treated with AGL, but not LPS (n=4).“X”: animals treated with FIN, but not LPS (n=4). FIG. 2D: Filleddiamonds: animals treated only with LPS (n=16) (data as in FIGS. 2A andB). Filled squares: animals treated with LPS and AGL (n=6). Hollowtriangles: animals treated with LPS and FIN (n=6).

FIGS. 3A-C. FIGS. 3A-3C are graphs depicting metabolomic analyses ofoxylipids and prostaglandins, revealing that there are significantdifferences in animals treated with sEH inhibitors and with an sEHinhibitor and a steroid synthesis inhibitor. For each Figure, the Y axisshows a scale in ng/mL for the bars set forth in the Figure, while the Xaxis shows the amounts of various metabolites, as indicated below theaxis. (In FIG. 3A, the bar presenting the results for the metabolite6-keto-PGF1a for one group of animals exceeded the scale, as shown by abreak in the bar and the statement of the result in ng/mL about thebar). FIG. 3A. This graph shows the amounts of the sum of thevic-dihydroxyeicosatrienoic acids (“DHETs”), the sum of the EETs,6-keto-PGF1a, PGF2a, and PGE2 in untreated (“naïve”) animals, in animalstreated with lipopolysaccharide (“LPS”), in animals treated with LPS andan inhibitor of sEH referred to as AEPU, and in animals treated withLPS, AEPU, and aminogluthetimide (“AGL”) FIG. 3B. This graph shows therelative amounts of the sum of the metabolite DiHOMEs (diols oflinoleate epoxide), the sum of the metabolites EpOMEs (linoleic acidmono-epoxides) and of the metabolite thromboxane B₂ (“TxB2”) in the fourgroups of animals described with regard to FIG. 3A. FIG. 3C. This graphshows the amounts of the sum of the hydroxyoctadecadienoic acids(“HODEs”) and the sum of the hydroxyeicosatetraenoic acids (“HETEs”) inthe four groups of animals described with regard to FIG. 3A. All threegraphs: bars with lines rising from left to right (e.g., in FIG. 3A, the“sum of DHETs”) present results for untreated (naïve) animals, bars withlines falling from left to right present results for animals treatedwith LPS, bars with cross-hatching present results for the animalstreated with LPS and the sEH inhibitor AEPU, and bars with horizontallines present the results for animals treated with LPS, AEPU, and thesteroid synthesis inhibitor aminogluthetimide (“AGL”). The metaboliteschosen for study show the effect on different pathways by whicharachidonic acid is metabolized. ΣEpOMEs and ΣDiHOMEs are indicators ofthe P450 pathway, HETEs are an indicator of how much arachidonic acid isgoing through the 5-lipoxygenase pathway and 6-keto-PGF_(1a), and PGE₂are indicators of the arachidonic acid metabolized by the cyclooxygenasepathway. 6-keto-PGF_(1a) and TXB₂ are stable metabolites of PGI₂ andthromboxane A₂ which have been implicated in increased risk for strokeand heart attack. HODEs are lipoxygenase-derived fatty acid metabolites.

FIG. 4. FIG. 4 is a graph of in vivo data showing that analgesia inducedby the action of sEH inhibitors is not blocked by steroid receptorantagonists. The Y axis shows hindpaw thermal withdrawal latencies(“TWL”) as a percentage of TWL prior to any treatment (“Baseline”). Thebars on X axis shows the result of testing using the agent or agentslisted below the bar. LPS: lipopolysaccharide. AEPU: sEH inhibitor.Tamoxifen is an estrogen receptor antagonist. Mifepristone is aglucocorticoid receptor antagonist. Nilutamide is an androgen receptorantagonist. Aminoglutethimide is a general steroid synthesis inhibitor.Finasteride is a 5 alpha reductase inhibitor that acts as a specificsteroid synthesis inhibitor. The line at the bottom of the Figure underwhich is stated “LPS+AEPU+” indicates that the bars above that linereflect the results of studies in which the animals were treated withLPS, AEPU, and the antagonist listed over the line.

FIG. 5. FIG. 5 is a graph of in vivo data showing that analgesia inducedby the action of sEH inhibitors is blocked by an antagonist of thecannabinoid receptor CB₂, but not by an antagonist of the cannabinoidreceptor CB₁. The Y axis shows hindpaw thermal withdrawal latencies(“TWL”) as a percentage of TWL prior to any treatment (“Baseline”). Thebars on X axis shows the result of testing using the agent or agentslisted below the bar at Baseline and two hours post administration oflipopolysaccharide (“LPS”). 950: sEH inhibitor compound 950. AM630:iodopravadoline, a CB₂ antagonist. AM251:N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methy1-1H-pyrazole-3-carboxamide, a CB₁ antagonist. + sign: shows experimentin which agent on corresponding horizontal line is present. − sign:shows experiment in which agent on corresponding horizontal line isabsent. Line above bar shows error range. First pair of bars showscontrol experiment in which LPS is not administered.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The enzyme “soluble epoxide hydrolase” (“sEH”) acts on an importantbranch of the arachidonic acid pathway degrading anti-inflammatory andanalgesic metabolites. cis-Epoxyeicosatrienoic acids” (“EETs”) arebiomediators synthesized by cytochrome P450 epoxygenases, and arehydrolyzed by sEH into the corresponding diols, which arepro-inflammatory.

Surprisingly, it has now been discovered that EETs bind to thecannabinoid CB₂ receptor, peripheral benzodiazepine receptor (“PBR”),neurokinin NK₂ receptor, and dopamine D₃ receptor. The binding dataalone did not, however, reveal whether EETs acted as agonists orantagonists of the biological functions of the receptors or would blockendogenous ligands of the receptors from reaching them, therebypreventing normal activation or antagonism of the receptors.

Surprisingly, the in vivo studies reported herein show that EETs act asagonists for PBR and for CB₂ receptors. Further, the in vitrocompetitive binding assays reported herein show that EETs displace knownhigh affinity ligands of the PBR and CB₂.

The findings reported herein show the pharmacological effect ofincreasing EETs in activating PBR activity. Persons of skill willtherefore appreciate that sEHIs, which are known to result in increasedlevels of EETs, and EETs themselves, will activate PBR activity whenadministered, and are therefore useful for treating conditions in whichmodulating (and specifically, increasing) the activity of PBR reduces oreliminates symptoms. Similarly, the assays reported herein show thepharmacological effect of increasing EETs in activating CB₂ activity.Persons of skill will therefore appreciate and expect that sEHIs, whichare known to result in increased levels of EETs, and EETs themselves,will activate CB₂ activity when administered, and are therefore usefulfor treating conditions in which increasing CB₂ activity reduces oreliminates symptoms.

The recognition that EETs binds to these molecular receptors permits theuse of EETs (and analogs of EETs that are not susceptible or are lesssusceptible to hydrolysis by sEH) to address conditions for which it wasnot previously known EETs could be used. Further, since theadministration of inhibitors of sEH increases the levels of EETs presentin the body, inhibitors of sEH can also be administered, alone or incombination with EETs, to increase EETs levels and therefore to addressthese conditions (for convenience, inhibitors of sEH are sometimesalternatively referred to herein as “sEHI”).

A selective CB₂ agonist has been shown to prevent the growth of gliomathrough a CB₂ dependant mechanism. In addition CB₂ receptors are knownto modulate peripheral nociceptive transmission. Further, a relationshipbetween cell proliferation and PBR expression has been observed in humanastrocytomas and breast cancer cell lines and PBR expression isupregulated in many types of cancer. Similarly, PBR ligands induce invitro inhibition of cancer cell proliferation and modulatesteroidogenesis. The activation of PBR receptors reduces proliferationthrough several mechanisms, such as that described Carrier et al.,Inhibition of an equilibrative nucleoside transporter by cannabidiol: Amechanism of cannabinoid immunosuppression, Proc Natl Acad Sci,103:7895-7900 (2006). Further, PBR ligands combined with cytotoxicagents have an anti-tumor effect in in vivo models. Since the studiesreported herein reveal that EETs are agonists of both the PBR and theCB2 receptors, it is expected that they will work through bothmechanisms to slow or prevent proliferation of glioma, astrocytoma andbreast cancer cells, as well as other cancer cell types in which PBRexpression is upregulated. Accordingly, administration of EETs,inhibitors of sEH, or both, can be administered to reduce the rate ofgrowth of glioma cells, astrocytoma cells, and breast cancer cells, andother malignant tumor cells expressing PBR, and especially those inwhich PBR expression is upregulated.

Further, agonists of PBR are known to act as anxiolytics. This ispresumably because of their ability to increase the acute synthesis ofneurosteroids such as allopregnanolone. sEHI and EETs are thereforeexpected to act as anxiolytics to reduce symptoms of anxiety. Withoutwishing to be bound by theory, this is expected to be through modulatingthe endogenous neurosteroid tone. Since administration of inhibitors ofsEH to an individual increases the level of EETs in the individualavailable to bind to the PBR during the period the inhibitor is presentand active in the individual, inhibitors of sEH are also expected to actas anxiolytics. Additionally, EETs, inhibitors of sEH, or both should beuseful for other indications in which anxiolytics are useful, includingas a premedication for inducing sedation, anxiolysis or amnesia prior tocertain medical procedures (e.g. endoscopy), as a means for reducingpanic attacks, and states of agitation, as a treatment for statusepilepticus, as adjunctive treatment of other forms of epilepsy, forreducing symptoms of alcohol and opiate withdrawal, for reducinginsomnia, and for initial management of mania, together with first linedrugs like lithium, valproate or other antipsychotics.

Since peripheral benzodiazepine receptors affect the rate ofsteroidogenesis, EETs or sEHI, or both, can be administered to affectthe rate of steroidogenesis. In particular, EETs, or sEHI, or both, canbe administered to reduce serum cholesterol levels. The EETs or sEHI, orboth, can be used alone or in conjunction with one or more statins toaugment the effect of the statin or statins.

Peripheral benzodiazepine receptors can also be targeted by EETs toprotect cells against oxygen radical damage. PER ligands are known todecrease UV damage to cells and tissues. Accordingly, it is expectedthat inhibitors of sEHI and EETs can be administered to reduce UV damageand oxygen radical damage to cells. In some embodiments, EETs orinhibitors of sEH are administered systemically to protect cells againstoxygen radical damage. Persons of skill are well aware of the potentialfor skin damage posed by prolonged exposure of skin to sunlight. In someembodiments, EETs or inhibitors of sEH are administered topically, forexample by being mixed into a lotion, cream or other base suitable fortopical administration, to reduce UV damage in skin exposed to sunlight.Conveniently, the cream or other base suitable for topicaladministration also contains a sunscreen or sunblock, such asoxybenzone, avobenzone, a cinnamate, octyl methoxycinnamate (OMC),ethylhexyl p-methoxycinnamate, a salicylate, octyl salicylate (OCS),para-aminobenzoic acid (PABA), padimate-O, octyl dimethyl paba,octocrylene, zinc oxide, titanium dioxide, benzophenone, orbenzophenone-3. Sunscreens and sunblocks typically work by physicallyblocking or absorbing UV radiation whereas, as noted, EETs andinhibitors of sEH reduce UV damage. The two methods of protecting skinare therefore complementary and the combination of the two types ofagent is expected to have at least additive, and possibly synergistic,effects in protecting skin. For example, EETs, inhibitors of sEH, orboth can be administered to reduce the effect of photo-aging (aging ofskin because of UV damage) and to reduce the likelihood of developingskin cancer due to repeated exposure to UV light. Since exposure toionizing radiation is also believed to result in part from damage byoxygen radicals, EETs, inhibitors of sEH, or both, can be used topicallyon persons undergoing radiation therapy, particularly of the head andneck, to reduce incidental damage to the skin during the exposure to theradiation.

Neurokinin A (“NKA”) and its receptor NK₂ have a known role inmodulating gastric motility. In contrast to the PBR and CB₂ receptors,however, where it is activation of the receptor that results inalleviating symptoms of the conditions listed above, for the NK₂receptor it is reducing the activity of the receptor that is associatedwith alleviating symptoms therapeutically useful. For example, anantagonist of NKA activity is currently in Phase II clinical trials forirritable bowel syndrome (“IBS”), and the relationship between theactivation of the NK₂ receptor and symptoms of IBS is well establishedin the art. The studies reported herein show that EETs displace ligandsfrom the NK₂ receptor. The ability of EETs to displace endogenousligands that would otherwise activate the receptor results indownregulating NK₂ receptor activity. Thus, EETs act as antagonists ofendogenous NK₂ ligands and can be used to reduce symptoms of conditionsthat result from or are aggravated by, NK₂ receptor activation,including IBS. The administration of EETs or sEHI, or both, to personssuffering from IBS is therefore expected to reduce those symptoms.

The studies in the Examples report the results of both in vitro and invivo assays. First, as shown in FIG. 1, an in vitro assay using the highaffinity PBR ligand PK 111951-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide, a powerfulPBR ligand. (See, Langer and Arbilla, Fund Clin Pharmacol 2(3):159-70(1988)). The 5,6, 11,12, and 14,15 EETs all showed the ability tocompletely inhibit the binding of PK11195 at millimolar concentrations,while an EETs-me mixture inhibited PK11195 binding at millimolarconcentrations in a dose-dependent manner, while a mixture ofEETs-methylesters inhibited PK11195 binding more potently than anyindividual EET. The Kd of PK 11195 is 2.7 nM, while EETs displaced thishigh affinity ligand with an IC₅₀ of 4.6 μM for the EET me mixture.

The in vitro assays showed that EETs bind to PBR, but not whether EETsact as agonists or as antagonists of the receptor, or simply block thebinding of other ligands which may have one of these activities. Todetermine what effect, if any, EETs have on the PBR, in vivo assays wereperformed. It is known that peripheral benzodiazepine receptors affectthe rate of steroidogenesis. We have previously found that inhibitors ofsEH have an effect as analgesics using a well accepted animal model formeasuring analgesia. We hypothesized that sEHI-elicited analgesia wasinduced through action on the PBR, and realized we could determinewhether determine whether EETs acted as an agonist of PBR, as anantagonist, or as neither, by co-administering inhibitors of sEH andcompounds that are inhibitors of steroid synthesis and seeing if theyblocked sEH-elicited analgesia. Steroid synthesis inhibitors havepreviously been shown to be antagonists of PBR. See, e.g., Papadopoulos,et al., Peripheral-type benzodiazepine receptor in neurosteroidbiosynthesis, neuropathology and neurological disorders, Neuroscience(138), p 749-756 (2006) and da Silva et al., Involvement of steroids inanti-inflammatory effects of PK11195 in a murine model of pleurisy.Mediators of Inflammation (13), p 93-103 (2004).

In vivo assays were conducted using two different inhibitors of steroidsynthesis. The steroid synthesis inhibitor aminogluthetimide (AGL),effectively blocks all steroid synthesis by inhibiting the first enzyme,P450scc, in the steroid synthesis pathway. When topically administeredto rats, AGL completely blocked the antihyperalgesic action of the sEHIAEPU in the LPS-elicited inflammatory pain model. See, FIG. 2A.Additionally, another inhibitor, finasteride, blocks 5α reductase andstops the steroid biosynthesis by blocking the conversion oftestosterone to dihydrotestosterone in case of steroids and theconversion of progesterone to allopregnanolone in case of neurosteroids.Finasteride also blocked the anti-hyperalgesic activity of AEPU. See,FIG. 2B. In contrast, however, in vivo assays employing a non-steroidalestrogen receptor antagonist, tamoxifen, a dualprogesterone/glucocorticoid receptor antagonist, mifepristone, anandrogen receptor antagonist, nilutamide, and an aldosterone receptorantagonist, spironolactone, showed that these antagonists did not haveany impact on the antihyperalgesic action of AEPU, indicating that sEHIsand/or EETs do not act through these steroid receptors. See, FIG. 4.

In vivo assays were also conducted to determine whether EETs act toactivate or to antagonize CB₂ receptor activity. We performed in vivoassays using antagonists of both CB₁ and CB₂, essentially blocking theactivity of these receptors, to determine the contribution ofcannabinoid receptor activation to sEHI attained analgesia. As shown inFIG. 5, a CB₁ antagonist, AM251(N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methy1-1H-pyrazole-3-carboxamide, see, e.g., Gatley et al., Eur J Pharmacol1996 Jul. 4; 307(3):331-8 (1996)) did not block sEHI-elicited analgesia,whereas a CB₂ antagonist, AM630 (iodopravadoline, an aminoalkylindole,see, e.g., Pertwee et al., Life Sci. 56(23-24):1949-55 (1995))completely blocked sEHI-elicited analgesia. These assays establishedthat if CB₂ receptors are blocked by a selective CB₂ antagonist such asAM630, sEHIs can not elicit analgesia and that CB₂ receptor activationis required for the analgesic activity of sEHIs. In contrast,elimination of the activity of CB₁ receptors had no impact on theanalgesic activity of sEHIs.

Medicaments of EETs can be made which can be administered by themselvesor in conjunction with one or more sEH inhibitors, or a medicamentcontaining one or more sEH inhibitors can optionally contain one or moreEETs. The EETs can be administered alone, or concurrently with a sEHinhibitor or following administration of a sEH inhibitor. It isunderstood that, like all drugs, sEH inhibitors have half lives definedby the rate at which they are metabolized by or excreted from the body,and that the sEH inhibitor will have a period following administrationduring which it will be present in amounts sufficient to be effective.If EETs administered after an sEH inhibitor are intended to beadministered while the sEH inhibition is still in effect, therefore, itis desirable that the EETs be administered during the period duringwhich the inhibitor will be present in amounts to be effective to delayhydrolysis of the EETs. Typically, in such a situation, the EET or EETswill be administered within 48 hours of administering an sEH inhibitor.More preferably, where the effect of the EET or EETs is intended to beenhanced by the effect of an sEHI, the EET or EETs are administeredwithin 24 hours of the inhibitor, and even more preferably within 12hours. In increasing order of desirability, the EET or EETs areadministered within 10, 8, 6, 4, 2, hours, 1 hour, or one half hourafter administration of the inhibitor. Most preferably, the EET or EETsare administered concurrently with the inhibitor. In some embodiments,the person being treated with the EET or EETs does not have one of thedisorders listed above as a condition which the subject being treatedwith an sEHI does not have. In some embodiments, the person beingtreated with the EET or EETs is not being treated for atherosclerosis,other inflammatory conditions, or other conditions in which inhibitionof adhesion molecule expression, particularly on endothelial cells, isdesirable.

In some embodiments, the sEH inhibitor may be a nucleic acid, such as asmall interfering RNA (siRNA) or a micro RNA (miRNA), which reducesexpression of a gene encoding sEH. Optionally, EETs may be administeredin combination with such a nucleic acid. Typically, a study willdetermine the time following administration of the nucleic acid before adecrease is seen in levels of sEH. The EET or EETs are typically thenadministered at a time calculated to be after expression of the nucleicacid has resulted in a decrease in sEH levels.

Patients Who can Benefit from Use of EETs or sEHI or Both

In some embodiments of the invention, the person being treated withEETs, sEHI, or both, does not have hypertension or is not currentlybeing treated with an anti-hypertension agent that is an inhibitor ofsEH. In some embodiments, the person being treated does not haveinflammation or, if he or she has inflammation, has not been treatedwith an sEH inhibitor as an anti-inflammatory agent. In some preferredembodiments, the person is being treated for inflammation but by ananti-inflammatory agent, such as a steroid, that is not an inhibitor ofsEH. Whether or not any particular anti-inflammatory oranti-hypertensive agent is also an sEH inhibitor can be readilydetermined by standard assays, such as those taught in U.S. Pat. No.5,955,496.

In some embodiments, the patient's disease or condition is not caused byan autoimmune disease or a disorder associated with a T-lymphocytemediated immune function autoimmune response. In some embodiments, thepatient does not have a pathological condition selected from type 1 ortype 2 diabetes, insulin resistance syndrome, atherosclerosis, coronaryartery disease, angina, ischemia, ischemic stroke, Raynaud's disease, orrenal disease. In some embodiments, the patient is not a person withdiabetes mellitus whose blood pressure is 130/80 or less, a person withmetabolic syndrome whose blood pressure is less than 130/85, a personwith a triglyceride level over 215 mg/dL, or a person with a cholesterollevel over 200 mg/dL or is a person with one or more of these conditionswho is not taking an inhibitor of sEH. In some embodiments, the patientdoes not have an obstructive pulmonary disease, an interstitial lungdisease, or asthma. In some embodiments, the patient is not alsocurrently being treated with an inhibitor of one or more enzymesselected from the group consisting of cyclo-oxygenase (“COX”)-1, COX-2,and 5-lipoxygenase (“5-LOX”), or 5-lipoxygenase activating protein(“FLAP”). It is noted that many people take a daily low dose of aspirin(e.g., 81 mg) to reduce their chance of heart attack, or take anoccasional aspirin to relieve a headache. It is not contemplated thatpersons taking low dose aspirin to reduce the risk of heart attack wouldordinarily take that aspirin in combination with an EET or sEHI topotentiate that effect. It is also not contemplated that persons takingan occasional aspirin or ibuprofen tablet to relieve a headache or otherepisodic minor aches or pain would ordinarily take that tablet incombination with an EET or sEHI to potentiate that pain relief, asopposed to persons seeking relief for chronic pain from arthritis orother conditions requiring significant pain relief over an extendedperiod. In some embodiments, therefore, the patient being treated by themethods of the invention may have taken an inhibitor of COX-1, COX-2, or5-LOX in low doses, or taken such an inhibitor on an occasional basis torelieve an occasional minor ache or pain. In some embodiments, thepatient does not have dilated cardiomyopathy or arrhythmia. In someembodiments, the patient is not using EETs or sEHI topically for painrelief. In some embodiments, the patient is not administering EETs orsEHI topically to the eye to relieve, for example, dry eye syndrome orintraocular pressure. In some embodiments, the patient does not haveglaucoma or is being treated for glaucoma with agents that do not alsoinhibit sEH.

DEFINITIONS

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; amino acid sequencesare written left to right in amino to carboxy orientation. The headingsprovided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety. Terms not defined herein have their ordinary meaning asunderstood by a person of skill in the art.

“cis-Epoxyeicosatrienoic acids” (“EETs”) are biomediators synthesized bycytochrome P450 epoxygenases. As discussed further in a separate sectionbelow, while the use of unmodified EETs is the most preferred,derivatives of EETs, such as amides and esters (both natural andsynthetic), EETs analogs, and EETs optical isomers can all be used inthe methods of the invention, both in pure form and as mixtures of theseforms. For convenience of reference, the term “EETs” as used hereinrefers to all of these forms unless otherwise required by context.

“Epoxide hydrolases” (“EH;” EC 3.3.2.3) are enzymes in the alpha betahydrolase fold family that add water to 3-membered cyclic ethers termedepoxides. The addition of water to the epoxides results in thecorresponding 1,2-diols (Hammock, B. D. et al., in ComprehensiveToxicology Biotransformation (Elsevier, New York), pp. 283-305 (1997);Oesch, F. Xenobiotica 3:305-340 (1972)). Four principal EH's are known:leukotriene epoxide hydrolase, cholesterol epoxide hydrolase, microsomalEH (“mEH”), and soluble EH (“sEH,” previously called cytosolic EH). Theleukotriene EH acts on leukotriene A4, whereas the cholesterol EHhydrates compounds related to the 5,6-epoxide of cholesterol. Themicrosomal epoxide hydrolase metabolizes monosubstituted,1,1-disubstituted, cis-1,2-disubstituted epoxides and epoxides on cyclicsystems to their corresponding diols. Because of its broad substratespecificity, this enzyme is thought to play a significant role inameliorating epoxide toxicity. Reactions of detoxification typicallydecrease the hydrophobicity of a compound, resulting in a more polar andthereby excretable substance.

“Soluble epoxide hydrolase” (“sEH”) is an epoxide hydrolase which inmany cell types converts EETs to dihydroxy derivatives calleddihydroxyeicosatrienoic acids (“DHETs”). The cloning and sequence of themurine sEH is set forth in Grant et al., J. Biol. Chem.268(23):17628-17633 (1993). The cloning, sequence, and accession numbersof the human sEH sequence are set forth in Beetham et al., Arch.Biochem. Biophys. 305(1):197-201 (1993). The amino acid sequence ofhuman sEH is SEQ ID NO.:1, while the nucleic acid sequence encoding thehuman sEH is SEQ ID NO.:2. (The sequence set forth as SEQ ID NO.:2 isthe coding portion of the sequence set forth in the Beetham et al. 1993paper and in the NCBI Entrez Nucleotide Browser at accession numberL05779, which include the 5′ untranslated region and the 3′ untranslatedregion.) The evolution and nomenclature of the gene is discussed inBeetham et al., DNA Cell Biol. 14(1):61-71 (1995). Soluble epoxidehydrolase represents a single highly conserved gene product with over90% homology between rodent and human (Arand et al., FEBS Lett.,338:251-256 (1994)). Soluble EH is only very distantly related to mEHand hydrates a wide range of epoxides not on cyclic systems. In contrastto the role played in the degradation of potential toxic epoxides bymEH, sEH is believed to play a role in the formation or degradation ofendogenous chemical mediators. Unless otherwise specified, as usedherein, the terms “soluble epoxide hydrolase” and “sEH” refer to humansEH.

Unless otherwise specified, as used herein, the term “sEH inhibitor”(also abbreviated as “sEHI”) refers to an inhibitor of human sEH.Preferably, the inhibitor does not also inhibit the activity ofmicrosomal epoxide hydrolase by more than 25% at concentrations at whichthe inhibitor inhibits sEH by at least 50%, and more preferably does notinhibit mEH by more than 10% at that concentration. For convenience ofreference, unless otherwise required by context, the term “sEHinhibitor” as used herein encompasses prodrugs which are metabolized toactive inhibitors of sEH. Further for convenience of reference, andexcept as otherwise required by context, reference herein to a compoundas an inhibitor of sEH includes reference to derivatives of thatcompound (such as an ester of that compound) that retain activity as ansEH inhibitor.

By “physiological conditions” is meant an extracellular milieu havingconditions (e.g., temperature, pH, and osmolarity) which allows for thesustenance or growth of a cell of interest.

“Micro-RNA” (“miRNA”) refers to small, noncoding RNAs of 18-25 nt inlength that negatively regulate their complementary mRNAs at theposttranscriptional level in many eukaryotic organisms. See, e.g.,Kurihara and Watanabe, Proc Natl Acad Sci USA 101(34):12753-12758(2004). Micro-RNA's were first discovered in the roundworm C. elegans inthe early 1990s and are now known in many species, including humans. Asused herein, it refers to exogenously administered miRNA unlessspecifically noted or otherwise required by context.

Neurokinin Receptors

Neurokinins are a family of regulatory peptides that are widelydistributed throughout the mammalian body where they are known to act asneurotransmitters in both the central and peripheral nervous systems. Inthe periphery, neuroknin receptors are mostly found incapsaicin-sensitive sensory nerves, which are now accepted not only torelay information to the central nervous system, but also to releasepeptide neurotransmitters from the efferent terminals; this release canbring about effects in surrounding tissues. The mammalian tachykininsinclude substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)which preferentially act at three G-protein-linked receptors termed NK₁,NK₂ and NK₃ respectively, though at high concentrations they can act atall three receptors. Activation of the neurokinin receptors can lead toa wide variety of biological actions such as smooth muscle contraction,vasodilation, secretion, neurogenic inflammation and activation of theimmune system. One of the known roles of NKA and its receptor NK₂ is inmodulating gastric motility. The Neurokinin NK₂ receptor is beingtargeted by several pharmaceutical companies for treatment ofgastrointestinal disorders. For example, the Menarini Group (Florence,Italy) has a NK₂ antagonist, Nepadutant (a glycosylated bicyclicpeptide) in Phase II clinical trials for bronchial hyperactivity andirritable bowel syndrome (IBS). Hyperalgesia is due to sensitization ofsensory receptors or nociceptors. Visceral hyperalgesia has beenrecognized as the main pathophysiological event underlying IBS symptoms.The proposed mechanism of action of this compound is that in animalmodels of IBS it corrects colon visceral hyperalgesia.

Cannabinoid Receptors

Cannabinoids, the active components of Cannabis saliva, and theirderivatives, exert a wide spectrum of central and peripheral actions,such as analgesia, anticonvulsion, anti-inflammation, and alleviation ofboth intraocular pressure and emesis. Two different cannabinoidreceptors have been characterized and cloned from mammalian tissues, CB₁and CB₂. CB₁ is expressed primarily in the central nervous system,whereas CB₂ is expressed primarily in cells of the immune system and isabsent in neurons of the central nervous system. Cannabinoid agonistssuppress nociceptive transmission and inhibit pain-related behavior inanimal models of acute and persistent nociception. CB₂-selectiveagonists fail to elicit centrally mediated cannabimimetic effects suchas hypothermia, catalepsy, and hypoactivity and are unlikely to bepsychoactive or addictive. Activation of CB₂ on non-neuronal cells ininflamed tissue is postulated to suppress the release of inflammatorymediators implicated in nociceptor sensitization. The recent developmentof selective agonists and antagonists for CB₂ has provided thepharmacological tools necessary to evaluate the role of CB₂ inmodulating persistent nociception. CB₂-selective agonists have recentlybeen shown to induce antinociception in models of acute, inflammatory,and nerve injury-induced nociception. AM1241, a CB₂-selective agonist,exhibits 340-fold selectivity for CB₂ over CB₁. AM1241 also attenuatesneuropathic pain through a CB₂ mechanism that is not dependent upon CB₁.Another selective CB₂ agonist JWH-133 prevents the growth of gliomathrough a CB₂ dependant mechanism.

Peripheral Benzodiazepine Receptors

Two main functions of peripheral benzodiazepine receptors (“PBR”) havebeen described: a role in steroidogenesis and modulation of theapoptotic process. With respect to steroidogenesis, PBR bind cholesteroland mediates its transport from the outer to the inner mitochondrialmembranes. This translocation is the first and rate limiting step forsteroid synthesis. PBR activation results in an increase in pregnenoloneformation and the synthesis of downstream steroids. PBR are alsoinvolved in human cancer cell proliferation. A relationship between cellproliferation and PBR expression has been observed in human astrocytomasand breast cancer cell lines. Similarly, PBR ligands induce in vitroinhibition of cancer cell proliferation.

Turning to apoptosis, apoptosis (also referred to as “programmed celldeath”) is mainly under the control of mitochondria; and themitochondrial permeability transition pore plays a key role in thisregulation. Mitochondrial membrane permeabilization (“MMP”) therefore isa major check-point in the cascade of biochemical events leading to theinduction of programmed cell death. A number of apoptosis-inducingsignals induce MMP and anti-apoptotic proteins block this alteration.The loss of mitochondrial membrane integrity leads to a drop oftransmembrane potential and remodeling of mitochondrial ultra-structurethat allow the release of toxic intermembrane proteins into thecytoplasm such as cytochrome c. These apoptotic effectors are thenresponsible for the late events of the cell death process. The PTPtherefore appears to be a multiprotein complex whose molecular dynamicscould be influenced by several partners. PBR is one of these partnersand can therefore be used as a target in clinical and therapeuticapproaches. Numerous observations indicate that PBR participates in theregulation of apoptosis: (i) transfection-enforced overexpression of PBRattenuates apoptosis induced by oxygen radicals or ultraviolet light,(ii) permeabilized mitochondria release DBI that binds intactmitochondria and accelerates MMP induction throughout the cell, (iii)the myxoma poxvirus M11L protein inhibits host cell apoptosis via aphysical and functional interaction with PBR, and (iv) various PBRligands with nanomolar affinity for the receptor, such as Ro-4864 andPK11195, modulate cancer cell response to apoptosis-inducing signals.PBR ligand-induced enhancement of apoptosis clearly acts viamitochondrial targeting. PBR ligands combined with cytotoxic agents havean anti-tumor effect in in vivo models.

There is also evidence for a role played by PBR in regulation ofinflammation processes, as various in vivo mouse models of acuteinflammation have shown that PBR ligands inhibit inflammatory signs ofpleurisy, arthritis or lupus erythematosus. These effects are thought tooccur through (i) modulation of the human natural killer cell activity,(ii) induction of heat shock protein expression, (iii) modulation of theactivity of monocytes/macrophages and (iv) restoration of the apoptoticprocess in auto-immune components. Other functions of PBR includeregulation of ischemia-reperfusion injury via membrane biogenesis,protection of hematopoietic cells against oxygen radical damage, lipidfluidity of mitochondria, modulation of bronchomotor tone, erythroiddifferentiation, intracellular transport of heme and porphyrins.

Irritable Bowel Syndrome

Irritable bowel syndrome, or IBS, is considered one of the most commonreasons people see their doctor in the U.S., accounting for more thanone out of every 10 doctor visits. According to the National DigestiveDiseases Information Clearinghouse, of the National Institute ofDiabetes and Digestive and Kidney Diseases (NIDDK), IBS is a functionaldisorder that affects mainly the bowel. IBS is characterized byover-sensitivity of the nerves and muscles of the bowel, which typicallyresults in cramping, bloating, gas, diarrhea, and constipation. Inpersons with IBS, symptoms can be triggered by stress, exercise, andhormones, as well as by foods such as milk products, chocolate, alcohol,caffeine, carbonated drinks, and fatty foods. Since there is no cure,patients with IBS are usually treated to relieve symptoms, by dietchanges, medicine such as anti-spasmotics to slow bowel contractions,and stress relief. One medication, alosetron(5-methyl-2-[(4-methyl-1H-imidazol-5-yl)methyl]-3,4-dihydro-2H-pyrido[4,3-b]indol-1(5H)-one),a 5-HT₄ antagonist used to block serotonin activity in the intestinaltract, is currently only approved for use in women with IBS in whichdiarrhea predominates, but its use is sharply limited due to potentiallyserious side effects on the gastrointestinal tract. A second, tegaserod(1-{[5-(hydroxymethyl)-1H-indol-3-yl]methylideneamino}-2-pentyl-guanidine)is also a serotonin type 4 receptor (“5-HT₄”) partial agonist and isapproved for short-term use in women with IBS. Since EETs bind to adifferent receptor than do alosetron and tegaserod, the problemsassociated with the use of these agents, and with alosetron inparticular, are not expected with the uses and methods of the presentinvention.

Inhibitors of Soluble Epoxide Hydrolase

Scores of sEH inhibitors are known, of a variety of chemical structures.Derivatives in which the urea, carbamate, or amide pharmacophore (asused herein, “pharmacophore” refers to the section of the structure of aligand that binds to the sEH) is covalently bound to both an adamantaneand to a 12 carbon chain dodecane are particularly useful as sEHinhibitors. Derivatives that are metabolically stable are preferred, asthey are expected to have greater activity in vivo. Selective andcompetitive inhibition of sEH in vitro by a variety of urea, carbamate,and amide derivatives is taught, for example, by Morisseau et al., Proc.Natl. Acad. Sci. U.S. A, 96:8849-8854 (1999), which provides substantialguidance on designing urea derivatives that inhibit the enzyme.

Derivatives of urea are transition state mimetics that form a preferredgroup of sEH inhibitors. Within this group, N,N′-dodecyl-cyclohexyl urea(DCU), is preferred as an inhibitor, while N-cyclohexyl-N′-dodecylurea(CDU) is particularly preferred. Some compounds, such asdicyclohexylcarbodiimide (a lipophilic diimide), can decompose to anactive urea inhibitor such as DCU. Any particular urea derivative orother compound can be easily tested for its ability to inhibit sEH bystandard assays, such as those discussed herein. The production andtesting of urea and carbamate derivatives as sEH inhibitors is set forthin detail in, for example, Morisseau et al., Proc Natl Acad Sci (USA)96:8849-8854 (1999).

N-Adamantyl-N′-dodecyl urea (“ADU”) is both metabolically stable and hasparticularly high activity on sEH. (Both the 1- and the 2-adamantylureas have been tested and have about the same high activity as aninhibitor of sEH.) Thus, isomers of adamantyl dodecyl urea are preferredinhibitors. It is further expected that N,N′-dodecyl-cyclohexyl urea(DCU), and other inhibitors of sEH, and particularly dodecanoic acidester derivatives of urea, are suitable for use in the methods of theinvention. Preferred inhibitors include:

12-(3-Adamantan-1-yl-ureido)dodecanoic acid (AUDA),

12-(3-Adamantan-1-yl-ureido)dodecanoic acid butyl ester (AUDA-BE),

Adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy}pentyl]urea (compound 950,also referred to herein as “AEPU”), and

Another preferred group of inhibitors are piperidines. The followingTable sets forth some exemplar piperidines and their ability to inhibitsEH activity, expressed as the amount needed to reduce the activity ofthe enzyme by 50% (expressed as “IC₅₀”).

TABLE 1 IC₅₀ values for selected alkylpiperidine-based sEH inhibitors

n = 0 n = 1 Compound IC₅₀ (μM)^(a) Compound IC₅₀ (μM)^(a) R: H I 0.30 II4.2

3a 3.8 4.a 3.9

3b 0.81 4b 2.6

3c 1.2 4c 0.61

3d 0.01 4d 0.11 ^(a)As determined via a kinetic fluorescent assay.

A number of other sEH inhibitors which can be used in the methods andcompositions of the invention are set forth in co-owned applicationsPCT/US2004/010298 and U.S. Published Patent Application Publication2005/0026844.

U.S. Pat. No. 5,955,496 (the '496 patent) also sets forth a number ofsEH inhibitors which can be use in the methods of the invention. Onecategory of these inhibitors comprises inhibitors that mimic thesubstrate for the enzyme. The lipid alkoxides (e.g., the 9-methoxide ofstearic acid) are an exemplar of this group of inhibitors. In additionto the inhibitors discussed in the '496 patent, a dozen or more lipidalkoxides have been tested as sEH inhibitors, including the methyl,ethyl, and propyl alkoxides of oleic acid (also known as stearic acidalkoxides), linoleic acid, and arachidonic acid, and all have been foundto act as inhibitors of sEH.

In another group of embodiments, the '496 patent sets forth sEHinhibitors that provide alternate substrates for the enzyme that areturned over slowly. Exemplars of this category of inhibitors are phenylglycidols (e.g., S,S-4-nitrophenylglycidol), and chalcone oxides. The'496 patent notes that suitable chalcone oxides include 4-phenylchalconeoxide and 4-fluourochalcone oxide. The phenyl glycidols and chalconeoxides are believed to form stable acyl enzymes.

Additional inhibitors of sEH suitable for use in the methods of theinvention are set forth in U.S. Pat. Nos. 6,150,415 (the '415 patent)and 6,531,506 (the '506 patent). Two preferred classes of inhibitors ofthe invention are compounds of Formulas 1 and 2, as described in the'415 and '506 patents. Means for preparing such compounds and assayingdesired compounds for the ability to inhibit epoxide hydrolases are alsodescribed. The '506 patent, in particular, teaches scores of inhibitorsof Formula 1 and some twenty inhibitors of Formula 2, which were shownto inhibit human sEH at concentrations as low as 0.1 μM. Any particularinhibitor can readily be tested to determine whether it will work in themethods of the invention by standard assays. Esters and salts of thevarious compounds discussed above or in the cited patents, for example,can be readily tested by these assays for their use in the methods ofthe invention.

As noted above, chalcone oxides can serve as an alternate substrate forthe enzyme. While chalcone oxides have half lives which depend in parton the particular structure, as a group the chalcone oxides tend to haverelatively short half lives (a drug's half life is usually defined asthe time for the concentration of the drug to drop to half its originalvalue. See, e.g., Thomas, G., Medicinal Chemistry: an introduction, JohnWiley & Sons Ltd. (West Sussex, England, 2000)). Since the various usesof the invention contemplate inhibition of sEH over differing periods oftime which can be measured in days, weeks, or months, chalcone oxides,and other inhibitors which have a half life whose duration is shorterthan the practitioner deems desirable, are preferably administered in amanner which provides the agent over a period of time. For example, theinhibitor can be provided in materials that release the inhibitorslowly. Methods of administration that permit high local concentrationsof an inhibitor over a period of time are known, and are not limited touse with inhibitors which have short half lives although, for inhibitorswith a relatively short half life, they are a preferred method ofadministration.

In addition to the compounds in Formula 1 of the '506 patent, whichinteract with the enzyme in a reversible fashion based on the inhibitormimicking an enzyme-substrate transition state or reaction intermediate,one can have compounds that are irreversible inhibitors of the enzyme.The active structures such as those in the Tables or Formula 1 of the'506 patent can direct the inhibitor to the enzyme where a reactivefunctionality in the enzyme catalytic site can form a covalent bond withthe inhibitor. One group of molecules which could interact like thiswould have a leaving group such as a halogen or tosylate which could beattacked in an SN2 manner with a lysine or histidine. Alternatively, thereactive functionality could be an epoxide or Michael acceptor such asan α/β-unsaturated ester, aldehyde, ketone, ester, or nitrile.

Further, in addition to the Formula 1 compounds, active derivatives canbe designed for practicing the invention. For example, dicyclohexyl thiourea can be oxidized to dicyclohexylcarbodiimide which, with enzyme oraqueous acid (physiological saline), will form an activedicyclohexylurea. Alternatively, the acidic protons on carbamates orureas can be replaced with a variety of substituents which, uponoxidation, hydrolysis or attack by a nucleophile such as glutathione,will yield the corresponding parent structure. These materials are knownas prodrugs or protoxins (Gilman et al., The Pharmacological Basis ofTherapeutics, 7th Edition, MacMillan Publishing Company, New York, p. 16(1985)) Esters, for example, are common prodrugs which are released togive the corresponding alcohols and acids enzymatically (Yoshigae etal., Chirality, 9:661-666 (1997)). The drugs and prodrugs can be chiralfor greater specificity. These derivatives have been extensively used inmedicinal and agricultural chemistry to alter the pharmacologicalproperties of the compounds such as enhancing water solubility,improving formulation chemistry, altering tissue targeting, alteringvolume of distribution, and altering penetration. They also have beenused to alter toxicology profiles.

There are many prodrugs possible, but replacement of one or both of thetwo active hydrogens in the ureas described here or the single activehydrogen present in carbamates is particularly attractive. Suchderivatives have been extensively described by Fukuto and associates.These derivatives have been extensively described and are commonly usedin agricultural and medicinal chemistry to alter the pharmacologicalproperties of the compounds. (Black et al., Journal of Agricultural andFood Chemistry, 21(5):747-751 (1973); Fahmy et al, Journal ofAgricultural and Food Chemistry, 26(3):550-556 (1978); Jojima et al.,Journal of Agricultural and Food Chemistry, 31(3):613-620 (1983); andFahmy et al., Journal of Agricultural and Food Chemistry, 29(3):567-572(1981).)

Such active proinhibitor derivatives are within the scope of the presentinvention, and the just-cited references are incorporated herein byreference. Without being bound by theory, it is believed that suitableinhibitors of the invention mimic the enzyme transition state so thatthere is a stable interaction with the enzyme catalytic site. Theinhibitors appear to form hydrogen bonds with the nucleophiliccarboxylic acid and a polarizing tyrosine of the catalytic site.

In some embodiments, the sEH inhibitor used in the methods taught hereinis a “soft drug.” Soft drugs are compounds of biological activity thatare rapidly inactivated by enzymes as they move from a chosen targetsite. EETs and simple biodegradable derivatives administered to an areaof interest may be considered to be soft drugs in that they are likelyto be enzymatically degraded by sEH as they diffuse away from the siteof interest following administration. Some sEHI, however, may diffuse orbe transported following administration to regions where their activityin inhibiting sEH may not be desired. Thus, multiple soft drugs fortreatment have been prepared. These include but are not limited tocarbamates, esters, carbonates and amides placed in the sEHI,approximately 7.5 angstroms from the carbonyl of the centralpharmacophore. These are highly active sEHI that yield biologicallyinactive metabolites by the action of esterase and/or amidase. Groupssuch as amides and carbamates on the central pharmacophores can also beused to increase solubility for applications in which that is desirablein forming a soft drug. Similarly, easily metabolized ethers maycontribute soft drug properties and also increase the solubility.

In some embodiments, sEH inhibition can include the reduction of theamount of sEH. As used herein, therefore, sEH inhibitors can thereforeencompass nucleic acids that inhibit expression of a gene encoding sEH.Many methods of reducing the expression of genes, such as reduction oftranscription and siRNA, are known, and are discussed in more detailbelow.

Preferably, the inhibitor inhibits sEH without also significantlyinhibiting microsomal epoxide hydrolase (“mEH”). Preferably, atconcentrations of 500 μM, the inhibitor inhibits sEH activity by atleast 50% while not inhibiting mEH activity by more than 10%. Preferredcompounds have an IC₅₀ (inhibition potency or, by definition, theconcentration of inhibitor which reduces enzyme activity by 50%) of lessthan about 500 μM. Inhibitors with IC₅₀s of less than 500 μM arepreferred, with IC₅₀s of less than 100 μM being more preferred and, inorder of increasing preference, an IC50 of 50 μM, 40 μM, 30 μM, 25 μM,20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM or even less being stillmore preferred. Assays for determining sEH activity are known in the artand described elsewhere herein.

EETs

EETs, which are epoxides of arachidonic acid, are known to be effectorsof blood pressure, regulators of inflammation, and modulators ofvascular permeability. Hydrolysis of the epoxides by sEH diminishes thisactivity. Inhibition of sEH raises the level of EETs since the rate atwhich the EETs are hydrolyzed into dihydroxyeicosatrienoic acids(“DHETs”) is reduced.

It has long been believed that EETs administered systemically would behydrolyzed too quickly by endogenous sEH to be helpful. For example, inone prior report of EETs administration, EETs were administered bycatheters inserted into mouse aortas. The EETs were infused continuouslyduring the course of the experiment because of concerns over the shorthalf life of the EETs. See, Liao and Zeldin, International PublicationWO 01/10438 (hereafter “Liao and Zeldin”). It also was not known whetherendogenous sEH could be inhibited sufficiently in body tissues to permitadministration of exogenous EET to result in increased levels of EETsover those normally present. Further, it was thought that EETs, asepoxides, would be too labile to survive the storage and handlingnecessary for therapeutic use.

In studies from the laboratory of the present inventors, however, it hasbeen shown that systemic administration of EETs in conjunction withinhibitors of sEH had better results than did administration of sEHinhibitors alone. EETs were not administered by themselves in thesestudies since it was anticipated they would be degraded too quickly tohave a useful effect. Additional studies from the laboratory of thepresent inventors have since shown, however, that administration of EETsby themselves has had therapeutic effect. Without wishing to be bound bytheory, it is surmised that the exogenous EET overwhelms endogenous sEH,and allows EETs levels to be increased for a sufficient period of timeto have therapeutic effect. Thus, EETs can be administered without alsoadministering an sEHI to provide a therapeutic effect. Moreover, we havefound that EETs, if not exposed to acidic conditions or to sEH arestable and can withstand reasonable storage, handling andadministration.

In short, sEHI, EETs, or co-administration of sEHIs and of EETs, can beused in the methods of the present invention. In some embodiments, oneor more EETs are administered to the patient without also administeringan sEHI. In some embodiments, one or more EETs are administered shortlybefore or concurrently with administration of an sEH inhibitor to slowhydrolysis of the EET or EETs. In some embodiments, one or more EETs areadministered after administration of an sEH inhibitor, but before thelevel of the sEHI has diminished below a level effective to slow thehydrolysis of the EETs.

EETs useful in the methods of the present invention include 14,15-LET,8,9-EET and 11,12-EET, and 5,6 EETs. Preferably, the EETs areadministered as the methyl ester, which is more stable. Persons of skillwill recognize that the EETs are regioisomers, such as 8S,9R- and14R,15S-EET. 8,9-EET, 11,12-EET, and 14R,15S-EET, are commerciallyavailable from, for example, Sigma-Aldrich (catalog nos. E5516, E5641,and E5766, respectively, Sigma-Aldrich Corp., St. Louis, Mo.).

If desired, EETs, analogs, or derivatives that retain activity can beused in place of or in combination with unmodified EETs. Liao andZeldin, supra, define EET analogs as compounds with structuralsubstitutions or alterations in an EET, and include structural analogsin which one or more EET olefins are removed or replaced with acetyleneor cyclopropane groups, analogs in which the epoxide moiety is replacedwith oxitane or furan rings and heteroatom analogs. In other analogs,the epoxide moiety is replaced with ether, alkoxides,difluorocycloprane, or carbonyl, while in others, the carboxylic acidmoiety is replaced with a commonly used mimic, such as a nitrogenheterocycle, a sulfonamide, or another polar functionality. In preferredforms, the analogs or derivatives are relatively stable as compared toan unmodified EET because they are more resistant than an unmodified EETto sEH and to chemical breakdown. “Relatively stable” means the rate ofhydrolysis by sEH is at least 25% less than the hydrolysis of theunmodified EET in a hydrolysis assay, and more preferably 50% or morelower than the rate of hydrolysis of an unmodified EET. Liao and Zeldinshow, for example, episulfide and sulfonamide EETs derivatives. Amideand ester derivatives of EETs and that are relatively stable arepreferred embodiments. In preferred forms, the analogs or derivativeshave the biological activity of the unmodified EET regioisomer fromwhich it is modified or derived in binding to the CB2 or peripheral BZDreceptor. Whether or not a particular EET analog or derivative has thebiological activity of the unmodified EET can be readily determined byusing it in standard assays, such as radio-ligand competition assays tomeasure binding to the relevant receptor. As mentioned in the Definitionsection, above, for convenience of reference, the term “EETs” as usedherein refers to unmodified EETs, and EETs analogs and derivativesunless otherwise required by context.

In some embodiments, the EET or EETs are embedded or otherwise placed ina material that releases the EET over time. Materials suitable forpromoting the slow release of compositions such as EETs are known in theart. Optionally, one or more sEH inhibitors may also be placed in theslow release material.

Conveniently, the EET or EETs can be administered orally. Since EETs aresubject to degradation under acidic conditions, EETs intended for oraladministration can be coated with a coating resistant to dissolvingunder acidic conditions, but which dissolve under the mildly basicconditions present in the intestines. Suitable coatings, commonly knownas “enteric coatings” are widely used for products, such as aspirin,which cause gastric distress or which would undergo degradation uponexposure to gastric acid. By using coatings with an appropriatedissolution profile, the coated substance can be released in a chosensection of the intestinal tract. For example, a substance to be releasedin the colon is coated with a substance that dissolves at pH 6.5-7,while substances to be released in the duodenum can be coated with acoating that dissolves at pH values over 5.5. Such coatings arecommercially available from, for example, Rohm Specialty Acrylics (RohmAmerica LLC, Piscataway, N.J.) under the trade name “Eudragit®”. Thechoice of the particular enteric coating is not critical to the practiceof the invention.

Assays for Epoxide Hydrolase Activity

Any of a number of standard assays for determining epoxide hydrolaseactivity can be used to determine inhibition of sEH. For example,suitable assays are described in Gill, et al., Anal Biochem 131:273-282(1983); and Borhan, et al., Analytical Biochemistry 231:188-200 (1995)).Suitable in vitro assays are described in Zeldin et al., J. Biol. Chem.268:6402-6407 (1993). Suitable in vivo assays are described in Zeldin etal., Arch Biochem Biophys 330:87-96 (1996). Assays for epoxide hydrolaseusing both putative natural substrates and surrogate substrates havebeen reviewed (see, Hammock, et al. In: Methods in Enzymology, VolumeIII, Steroids and Isoprenoids, Part B, (Law, J. H. and H. C. Rifling,eds. 1985), Academic Press, Orlando, Fla., pp. 303-311 and Wixtrom etal., In: Biochemical Pharmacology and Toxicology, Vol. 1: MethodologicalAspects of Drug Metabolizing Enzymes, (Zakim, D. and D. A. Vessey, eds.1985), John Wiley & Sons, Inc., New York, pp. 1-93. Several spectralbased assays exist based on the reactivity or tendency of the resultingdiol product to hydrogen bond (see, e.g., Wixtrom, supra, and Hammock.Anal. Biochem. 174:291-299 (1985) and Dietze, et al. Anal. Biochem.216:176-187 (1994)).

The enzyme also can be detected based on the binding of specific ligandsto the catalytic site which either immobilize the enzyme or label itwith a probe such as dansyl, fluoracein, luciferase, green fluorescentprotein or other reagent. The enzyme can be assayed by its hydration ofEETs, its hydrolysis of an epoxide to give a colored product asdescribed by Dietze et al., 1994, supra, or its hydrolysis of aradioactive surrogate substrate (Borhan et al., 1995, supra). The enzymealso can be detected based on the generation of fluorescent productsfollowing the hydrolysis of the epoxide. Numerous method of epoxidehydrolase detection have been described (see, e.g., Wixtrom, supra).

The assays are normally carried out with a recombinant enzyme followingaffinity purification. They can be carried out in crude tissuehomogenates, cell culture or even in vivo, as known in the art anddescribed in the references cited above.

Other Means of Inhibiting sEH Activity

Other means of inhibiting sEH activity or gene expression can also beused in the methods of the invention. For example, a nucleic acidmolecule complementary to at least a portion of the human sEH gene canbe used to inhibit sEH gene expression. Means for inhibiting geneexpression using short RNA molecules, for example, are known. Amongthese are short interfering RNA (siRNA), small temporal RNAs (stRNAs),and micro-RNAs (miRNAs). Short interfering RNAs silence genes through amRNA degradation pathway, while stRNAs and miRNAs are approximately 21or 22 nt RNAs that are processed from endogenously encodedhairpin-structured precursors, and function to silence genes viatranslational repression. See, e.g., McManus et al., RNA, 8(6):842-50(2002); Morris et al., Science, 305(5688):1289-92 (2004); He and Hannon,Nat Rev Genet. 5(7):522-31 (2004).

“RNA interference,” a form of post-transcriptional gene silencing(“PTGS”), describes effects that result from the introduction ofdouble-stranded RNA into cells (reviewed in Fire, A. Trends Genet15:358-363 (1999); Sharp, P. Genes Dev 13:139-141 (1999); Hunter, C.Curr Biol 9:R440-R442 (1999); Baulcombe. D. Curr Biol 9:R599-R601(1999); Vaucheret et al. Plant J 16: 651-659 (1998)). RNA interference,commonly referred to as RNAi, offers a way of specifically inactivatinga cloned gene, and is a powerful tool for investigating gene function.

The active agent in RNAi is a long double-stranded (antiparallel duplex)RNA, with one of the strands corresponding or complementary to the RNAwhich is to be inhibited. The inhibited RNA is the target RNA. The longdouble stranded RNA is chopped into smaller duplexes of approximately 20to 25 nucleotide pairs, after which the mechanism by which the smallerRNAs inhibit expression of the target is largely unknown at this time.While RNAi was shown initially to work well in lower eukaryotes, formammalian cells, it was thought that RNAi might be suitable only forstudies on the oocyte and the preimplantation embryo.

In mammalian cells other than these, however, longer RNA duplexesprovoked a response known as “sequence non-specific RNA interference,”characterized by the non-specific inhibition of protein synthesis.

Further studies showed this effect to be induced by dsRNA of greaterthan about 30 base pairs, apparently due to an interferon response. Itis thought that dsRNA of greater than about 30 base pairs binds andactivates the protein PKR and 2′,5′-oligonucleotide synthetase(2′,5′-AS). Activated PKR stalls translation by phosphorylation of thetranslation initiation factors eIF2α, and activated 2′,5′-AS causes mRNAdegradation by 2′,5′-oligonucleotide-activated ribonuclease L. Theseresponses are intrinsically sequence-nonspecific to the inducing dsRNA;they also frequently result in apoptosis, or cell death. Thus, mostsomatic mammalian cells undergo apoptosis when exposed to theconcentrations of dsRNA that induce RNAi in lower eukaryotic cells.

More recently, it was shown that RNAi would work in human cells if theRNA strands were provided as pre-sized duplexes of about 19 nucleotidepairs, and RNAi worked particularly well with small unpaired 3′extensions on the end of each strand (Elbashir et al. Nature 411:494-498 (2001)). In this report, “short interfering RNA” (siRNA, alsoreferred to as small interfering RNA) were applied to cultured cells bytransfection in oligofectamine micelles. These RNA duplexes were tooshort to elicit sequence-nonspecific responses like apoptosis, yet theyefficiently initiated RNAi. Many laboratories then tested the use ofsiRNA to knock out target genes in mammalian cells. The resultsdemonstrated that siRNA works quite well in most instances.

For purposes of reducing the activity of sEH, siRNAs to the geneencoding sEH can be specifically designed using computer programs. Thecloning, sequence, and accession numbers of the human sEH sequence areset forth in Beetham et al., Arch. Biochem. Biophys. 305(1):197-201(1993). The amino acid sequence of human sEH (SEQ ID NO:1) and thenucleotide sequence encoding that amino acid sequence (SEQ ID NO.:2) areset forth in U.S. Pat. No. 5,445,956.

A program, siDESIGN from Dharmacon, Inc. (Lafayette, Colo.), permitspredicting siRNAs for any nucleic acid sequence, and is available on theWorld Wide Web at dharmacon.com. Programs for designing siRNAs are alsoavailable from others, including Genscript (available on the Web atgenscript.com/ssl-bin/app/rnai) and, to academic and non-profitresearchers, from the Whitehead Institute for Biomedical Research on theinternet by entering “http://” followed by“jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/.”

For example, using the program available from the Whitehead Institute,the following sEH target sequences and siRNA sequences can be generated:

1) (SEQ ID NO: 3) Target: CAGTGTTCATTGGCCATGACTGG (SEQ ID NO: 4)Sense-siRNA: 5′-GUGUUCAUUGGCCAUGACUTT-3′ (SEQ ID NO: 5) Antisense-siRNA:5′-AGUCAUGGCCAAUGAACACTT-3′ 2) (SEQ ID NO: 6) Target:GAAAGGCTATGGAGAGTCATCTG (SEQ ID NO: 7) Sense-siRNA:5′-AAGGCUAUGGAGAGUCAUCTT-3′ (SEQ ID NO: 8) Antisense-siRNA:5′-GAUGACUCUCCAUAGCCUUTT-3′ 3) (SEQ ID NO: 9) TargetAAAGGCTATGGAGAGTCATCTGC (SEQ ID NO: 10) Sense-siRNA:5′-AGGCUAUGGAGAGUCAUCUTT-3′ (SEQ ID NO: 11) Antisense-siRNA:5′-AGAUGACUCUCCAUAGCCUTT-3′ 4) (SEQ ID NO: 12) Target:CAAGCAGTGTTCATTGGCCATGA (SEQ ID NO: 13 Sense-siRNA:5′-AGCAGUGUUCAUUGGCCAUTT-3′ (SEQ ID NO: 14 Antisense-siRNA:5′-AUGGCCAAUGAACACUGCUTT-3′ 5) (SEQ ID NO: 15) Target:CAGCACATGGAGGACTGGATTCC (SEQ ID NO: 16) Sense-siRNA:5′-GCACAUGGAGGACUGGAUUTT-3′ (SEQ ID NO: 17) Antisense-siRNA:5′-AAUCCAGUCCUCCAUGUGCTT-3′

Alternatively, siRNA can be generated using kits which generate siRNAfrom the gene. For example, the “Dicer siRNA Generation” kit (catalognumber T510001, Gene Therapy Systems, Inc., San Diego, Calif.) uses therecombinant human enzyme “dicer” in vitro to cleave long double strandedRNA into 22 bp siRNAs. By having a mixture of siRNAs, the kit permits ahigh degree of success in generating siRNAs that will reduce expressionof the target gene. Similarly, the Silencer™ siRNA Cocktail Kit (RNaseIII) (catalog no. 1625, Ambion, Inc., Austin, Tex.) generates a mixtureof siRNAs from dsRNA using RNase III instead of dicer. Like dicer, RNaseIII cleaves dsRNA into 12-30 bp dsRNA fragments with 2 to 3 nucleotide3′ overhangs, and 5′-phosphate and 3′-hydroxyl termini. According to themanufacturer, dsRNA is produced using T7 RNA polymerase, and reactionand purification components included in the kit. The dsRNA is thendigested by RNase III to create a population of siRNAs. The kit includesreagents to synthesize long dsRNAs by in vitro transcription and todigest those dsRNAs into siRNA-like molecules using RNase III. Themanufacturer indicates that the user need only supply a DNA templatewith opposing T7 phage polymerase promoters or two separate templateswith promoters on opposite ends of the region to be transcribed.

The siRNAs can also be expressed from vectors. Typically, such vectorsare administered in conjunction with a second vector encoding thecorresponding complementary strand. Once expressed, the two strandsanneal to each other and form the functional double stranded siRNA. Oneexemplar vector suitable for use in the invention is pSuper, availablefrom OligoEngine, Inc. (Seattle, Wash.). In some embodiments, the vectorcontains two promoters, one positioned downstream of the first and inantiparallel orientation. The first promoter is transcribed in onedirection, and the second in the direction antiparallel to the first,resulting in expression of the complementary strands. In yet another setof embodiments, the promoter is followed by a first segment encoding thefirst strand, and a second segment encoding the second strand. Thesecond strand is complementary to the palindrome of the first strand.Between the first and the second strands is a section of RNA serving asa linker (sometimes called a “spacer”) to permit the second strand tobend around and anneal to the first strand, in a configuration known asa “hairpin.”

The formation of hairpin RNAs, including use of linker sections, is wellknown in the art. Typically, an siRNA expression cassette is employed,using a Polymerase III promoter such as human U6, mouse U6, or human H1.The coding sequence is typically a 19-nucleotide sense siRNA sequencelinked to its reverse complementary antisense siRNA sequence by a shortspacer. Nine-nucleotide spacers are typical, although other spacers canbe designed. For example, the Ambion website indicates that itsscientists have had success with the spacer TTCAAGAGA (SEQ ID NO:18).Further, 5-6 T's are often added to the 3′ end of the oligonucleotide toserve as a termination site for Polymerase III. See also, Yu et al., MolTher 7(2):228-36 (2003); Matsukura et al., Nucleic Acids Res 31(15):e77(2003).

As an example, the siRNA targets identified above can be targeted byhairpin siRNA as follows. To attack the same targets by short hairpinRNAs, produced by a vector (permanent RNAi effect), sense and antisensestrand can be put in a row with a loop forming sequence in between andsuitable sequences for an adequate expression vector to both ends of thesequence. The following are non-limiting examples of hairpin sequencesthat can be cloned into the pSuper vector:

1) Target: (SEQ ID NO: 19) CAGTGTTCATTGGCCATGACTGG Sense strand:(SEQ ID NO: 20) 5′-GATCCCCGTGTTCATTGGCCATGACTTTCAAGAGAAGTCATGGCCAATGAACACTTTTT-3′ Antisense strand: (SEQ ID NO: 21)5′-AGCTAAAAAGTGTTCATTGGCCATGACTTCTCTT GAAAGTCATGGCCAATGAACACGGG-3′ 2)Target: (SEQ ID NO: 22) GAAAGGCTATGGAGAGTCATCTG Sense strand:(SEQ ID NO: 23) 5′-GATCCCCAAGGCTATGGAGAGTCATCTTCAAGAGAGATGACTCTCCATAGCCTTTTTTT-3′ Antisense strand: (SEQ ID NO: 24)5′-AGCTAAAAAAAGGCTATGGAGAGTCATCTCTCTTGAA GATGACTCTCCATAGCCTTGGG-3′ 3)Target: (SEQ ID NO: 25) AAAGGCTATGGAGAGTCATCTGC Sense strand:(SEQ ID NO: 26) 5′-GATCCCCAGGCTATGGAGAGTCATCTTTCAAGAGAAGATGACTCTCCATAGCCTTTTTT-3′ Antisense strand: (SEQ ID NO: 27)5′-AGCTAAAAAAGGCTATGGAGAGTCATCATCTCTTGAAAGATGACTCT CCATAGCCTGGG-3′ 4)Target: (SEQ ID NO: 28) CAAGCAGTGTTCATTGGCCATGA Sense strand:(SEQ ID NO: 29) 5′-GATCCCCAGCAGTGTTCATTGGCCATTTCAAGAGAATGGCCAATGAACACTGCTTTTTT-3′ Antisense strand: (SEQ ID NO: 30)5′-AGCTAAAAAAGCAGTGTTCATTGGCCATTCTCTTGAAATG GCCAATGAACACTGCTGGG-3′ 5)Target: (SEQ ID NO: 31) CAGCACATGGAGGACTGGATTCC Sense strand(SEQ ID NO: 32) 5′-GATCCCCGCACATGGAGGACTGGATTTTCAAGAGAAATCCAGTCCTCCATGTGCTTTTT-3′ Antisense strand: (SEQ ID NO: 33)5′-AGCTAAAAAGCACATGGAGGACTGGATTTCTCTTGAAAA TCCAGTCCTCCATGTGCGGG-3′

In addition to siRNAs, other means are known in the art for inhibitingthe expression of antisense molecules, ribozymes, and the like are wellknown to those of skill in the art. The nucleic acid molecule can be aDNA probe, a riboprobe, a peptide nucleic acid probe, a phosphorothioateprobe, or a 2′-O methyl probe.

Generally, to assure specific hybridization, the antisense sequence issubstantially complementary to the target sequence. In certainembodiments, the antisense sequence is exactly complementary to thetarget sequence. The antisense polynucleotides may also include,however, nucleotide substitutions, additions, deletions, transitions,transpositions, or modifications, or other nucleic acid sequences ornon-nucleic acid moieties so long as specific binding to the relevanttarget sequence corresponding to the sEH gene is retained as afunctional property of the polynucleotide. In one embodiment, theantisense molecules form a triple helix-containing, or “triplex” nucleicacid. Triple helix formation results in inhibition of gene expressionby, for example, preventing transcription of the target gene (see, e.g.,Cheng et al., 1988, J. Biol. Chem. 263:15110; Ferrin and Camerini-Otero,1991, Science 354:1494; Ramdas et al., 1989, J. Biol. Chem. 264:17395;Strobel et al., 1991, Science 254:1639; and Rigas et al., 1986, Proc.Natl. Acad. Sci. U.S.A. 83:9591)

Antisense molecules can be designed by methods known in the art. Forexample, Integrated DNA Technologies (Coralville, Iowa) makes availablea program on the internet which can be found by entering http://,followed by biotools.idtdna.com/antisense/AntiSense.aspx, which willprovide appropriate antisense sequences for nucleic acid sequences up to10,000 nucleotides in length. Using this program with the sEH geneprovides the following exemplar sequences:

(SEQ ID NO: 34) 1) UGUCCAGUGCCCACAGUCCU (SEQ ID NO: 35)2) UUCCCACCUGACACGACUCU (SEQ ID NO: 36) 3) GUUCAGCCUCAGCCACUCCU(SEQ ID NO: 37) 4) AGUCCUCCCGCUUCACAGA (SEQ ID NO: 38)5) GCCCACUUCCAGUUCCUUUCC

In another embodiment, ribozymes can be designed to cleave the mRNA at adesired position. (See, e.g., Cech, 1995, Biotechnology 13:323; andEdgington, 1992, Biotechnology 10:256 and Hu et al., PCT Publication WO94/03596).

The antisense nucleic acids (DNA, RNA, modified, analogues, and thelike) can be made using any suitable method for producing a nucleicacid, such as the chemical synthesis and recombinant methods disclosedherein and known to one of skill in the art. In one embodiment, forexample, antisense RNA molecules of the invention may be prepared by denovo chemical synthesis or by cloning. For example, an antisense RNA canbe made by inserting (ligating) a sEH gene sequence in reverseorientation operably linked to a promoter in a vector (e.g., plasmid).Provided that the promoter and, preferably termination andpolyadenylation signals, are properly positioned, the strand of theinserted sequence corresponding to the noncoding strand will betranscribed and act as an antisense oligonucleotide of the invention.

It will be appreciated that the oligonucleotides can be made usingnonstandard bases (e.g., other than adenine, cytidine, guanine, thymine,and uridine) or nonstandard backbone structures to provides desirableproperties (e.g., increased nuclease-resistance, tighter-binding,stability or a desired Tm). Techniques for rendering oligonucleotidesnuclease-resistant include those described in PCT Publication WO94/12633. A wide variety of useful modified oligonucleotides may beproduced, including oligonucleotides having a peptide-nucleic acid (PNA)backbone (Nielsen et al., 1991, Science 254:1497) or incorporating2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioatenucleotides, phosphoramidates.

Proteins have been described that have the ability to translocatedesired nucleic acids across a cell membrane. Typically, such proteinshave amphiphilic or hydrophobic subsequences that have the ability toact as membrane-translocating carriers. For example, homeodomainproteins have the ability to translocate across cell membranes. Theshortest internalizable peptide of a homeodomain protein, Antennapedia,was found to be the third helix of the protein, from amino acid position43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology6:629-634 (1996). Another subsequence, the h (hydrophobic) domain ofsignal peptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al., J. Biol. Chem. 270:14255-14258(1995)). Such subsequences can be used to translocate oligonucleotidesacross a cell membrane. Oligonucleotides can be conveniently derivatizedwith such sequences. For example, a linker can be used to link theoligonucleotides and the translocation sequence. Any suitable linker canbe used, e.g., a peptide linker or any other suitable chemical linker.

More recently, it has been discovered that siRNAs can be introduced intomammals without eliciting an immune response by encapsulating them innanoparticles of cyclodextrin. Information on this method can be foundby entering “www.” followed by“nature.com/news/2005/050418/full/050418-6.html.”

In another method, the nucleic acid is introduced directly intosuperficial layers of the skin or into muscle cells by a jet ofcompressed gas or the like. Methods for administering nakedpolynucleotides are well known and are taught, for example, in U.S. Pat.No. 5,830,877 and International Publication Nos. WO 99/52483 and94/21797. Devices for accelerating particles into body tissues usingcompressed gases are described in, for example, U.S. Pat. Nos.6,592,545, 6,475,181, and 6,328,714. The nucleic acid may be lyophilizedand may be complexed, for example, with polysaccharides to form aparticle of appropriate size and mass for acceleration into tissue.Conveniently, the nucleic acid can be placed on a gold bead or otherparticle which provides suitable mass or other characteristics. Use ofgold beads to carry nucleic acids into body tissues is taught in, forexample, U.S. Pat. Nos. 4,945,050 and 6,194,389.

The nucleic acid can also be introduced into the body in a virusmodified to serve as a vehicle without causing pathogenicity. The viruscan be, for example, adenovirus, fowlpox virus or vaccinia virus.

miRNAs and siRNAs differ in several ways: miRNA derive from points inthe genome different from previously recognized genes, while siRNAsderive from mRNA, viruses or transposons, miRNA derives from hairpinstructures, while siRNA derives from longer duplexed RNA, miRNA isconserved among related organisms, while siRNA usually is not, and miRNAsilences loci other than that from which it derives, while siRNAsilences the loci from which it arises. Interestingly, miRNAs tend notto exhibit perfect complementarity to the mRNA whose expression theyinhibit. See, McManus et al., supra. See also, Cheng et al., NucleicAcids Res. 33(4):1290-7 (2005); Robins and Padgett, Proc Natl Acad SciUSA. 102(10:4006-9 (2005); Brennecke et al., PLoS Biol. 3(3):e85 (2005).Methods of designing miRNAs are known. See, e.g., Zeng et al., MethodsEnzymol. 392:371-80 (2005); Krol et al., J Biol Chem. 279(40):42230-9(2004); Ying and Lin, Biochem Biophys Res Commun. 326(3):515-20 (2005).

Therapeutic Administration

EETs and inhibitors of sEH can be prepared and administered in a widevariety of oral, parenteral and aerosol formulations. In some preferredforms, compounds for use in the methods of the present invention can beadministered by injection, that is, intravenously, intramuscularly,intracutaneously, subcutaneously, intraduodenally, or intraperitoneally,while in others, they are administered orally. The sEH inhibitor orEETs, or both, can also be administered by inhalation. Additionally, thesEH inhibitors, or EETs, or both, can be administered transdermally.Accordingly, the methods of the invention permit administration ofpharmaceutical compositions comprising a pharmaceutically acceptablecarrier or excipient and either a selected inhibitor or apharmaceutically acceptable salt of the inhibitor.

For preparing pharmaceutical compositions from sEH inhibitors, or EETs,or both, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier can be one or more substances which may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the active compound. Suitable carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution. Transdermal administration can beperformed using suitable carriers. If desired, apparatuses designed tofacilitate transdermal delivery can be employed. Suitable carriers andapparatuses are well known in the art, as exemplified by U.S. Pat. Nos.6,635,274, 6,623,457, 6,562,004, and 6,274,166.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The term “unit dosage form”, as used in the specification, refers tophysically discrete units suitable as unitary dosages for human subjectsand animals, each unit containing a predetermined quantity of activematerial calculated to produce the desired pharmaceutical effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular effect to beachieved and (b) the limitations inherent in the art of compounding suchan active material for use in humans and animals, as disclosed in detailin this specification, these being features of the present invention.

A therapeutically effective amount of the sEH inhibitor, or EETs, orboth, is employed in inhibiting cardiac arrhythmia or inhibiting orreversing cardiac hypertrophy or dilated cardiomyopathy. The dosage ofthe specific compound for treatment depends on many factors that arewell known to those skilled in the art. They include for example, theroute of administration and the potency of the particular compound. Anexemplary dose is from about 0.001 μM/kg to about 100 mg/kg body weightof the mammal.

EETs are unstable in acidic conditions, and can be converted to DHETs.To avoid conversion of orally administered EETs to DHETs under theacidic conditions present in the stomach, EETs can be administeredintravenously, by injection, or by aerosol. EETs intended for oraladministration can be encapsulated in a coating that protects the EETsduring passage through the stomach. For example, the EETs can beprovided with a so-called “enteric” coating, such as those used for somebrands of aspirin, or embedded in a formulation. Such enteric coatingsand formulations are well known in the art. In some formulations, theEETs, or a combination of the EETs and an sEH inhibitor are embedded ina slow-release formulation to facilitate administration of the agentsover time.

In another set of embodiments, an sEH inhibitor, one or more EETs, orboth an sEH inhibitor and an EET are administered by delivery to thenose or to the lung. Intranasal and pulmonary delivery are considered tobe ways drugs can be rapidly introduced into an organism. Devices fordelivering drugs intranasally or to the lungs are well known in the art.The devices typically deliver either an aerosol of an therapeuticallyactive agent in a solution, or a dry powder of the agent. To aid inproviding reproducible dosages of the agent, dry powder formulationsoften include substantial amounts of excipients, such aspolysaccharides, as bulking agents.

Detailed information about the delivery of therapeutically active agentsin the form of aerosols or as powders is available in the art. Forexample, the Center for Drug Evaluation and Research (“CDER”) of theU.S. Food and Drug Administration provides detailed guidance in apublication entitled: “Guidance for Industry: Nasal Spray and InhalationSolution, Suspension, and Spray Drug Products—Chemistry, Manufacturing,and Controls Documentation” (Office of Training and Communications,Division of Drug Information, CDER, FDA, July 2002). This guidance isavailable in written form from CDER, or can be found on-line by entering“http://www.” followed by “fda.gov/cder/guidance/4234fnl.htm”. The FDAhas also made detailed draft guidance available on dry powder inhalersand metered dose inhalers. See, Metered Dose Inhaler (MDI) and DryPowder Inhaler (DPI) Drug Products—Chemistry, Manufacturing, andControls Documentation, 63 Fed. Reg. 64270, (November 1998). A number ofinhalers are commercially available, for example, to administeralbuterol to asthma patients, and can be used instead in the methods ofthe present invention to administer the sEH inhibitor, EET, or acombination of the two agents to subjects in need thereof.

In some aspects of the invention, the sEH inhibitor, EET, or combinationthereof, is dissolved or suspended in a suitable solvent, such as water,ethanol, or saline, and administered by nebulization. A nebulizerproduces an aerosol of fine particles by breaking a fluid into finedroplets and dispersing them into a flowing stream of gas. Medicalnebulizers are designed to convert water or aqueous solutions orcolloidal suspensions to aerosols of fine, inhalable droplets that canenter the lungs of a patient during inhalation and deposit on thesurface of the respiratory airways. Typical pneumatic (compressed gas)medical nebulizers develop approximately 15 to 30 microliters of aerosolper liter of gas in finely divided droplets with volume or mass mediandiameters in the respirable range of 2 to 4 micrometers. Predominantly,water or saline solutions are used with low solute concentrations,typically ranging from 1.0 to 5.0 mg/mL.

Nebulizers for delivering an aerosolized solution to the lungs arecommercially available from a number of sources, including the AERx™(Aradigm Corp., Hayward, Calif.) and the Acorn II® (Vital Signs Inc.,Totowa, N.J.).

Metered dose inhalers are also known and available. Breath actuatedinhalers typically contain a pressurized propellant and provide ametered dose automatically when the patient's inspiratory effort eithermoves a mechanical lever or the detected flow rises above a presetthreshold, as detected by a hot wire anemometer. See, for example, U.S.Pat. Nos. 3,187,748; 3,565,070; 3,814,297; 3,826,413; 4,592,348;4,648,393; 4,803,978; and 4,896,832.

The formulations may also be delivered using a dry powder inhaler (DPI),i.e., an inhaler device that utilizes the patient's inhaled breath as avehicle to transport the dry powder drug to the lungs. Such devices aredescribed in, for example, U.S. Pat. Nos. 5,458,135; 5,740,794; and5,785,049. When administered using a device of this type, the powder iscontained in a receptacle having a puncturable lid or other accesssurface, preferably a blister package or cartridge, where the receptaclemay contain a single dosage unit or multiple dosage units.

Other dry powder dispersion devices for pulmonary administration of drypowders include those described in Newell, European Patent No. EP129985; in Hodson, European Patent No. EP 472598, in Cocozza, EuropeanPatent No. EP 467172, and in Lloyd, U.S. Pat. Nos. 5,522,385; 4,668,281;4,667,668; and 4,805,811. Dry powders may also be delivered using apressurized, metered dose inhaler (MDI) containing a solution orsuspension of drug in a pharmaceutically inert liquid propellant, e.g.,a chlorofluorocarbon or fluorocarbon, as described in U.S. Pat. Nos.5,320,094 and 5,672,581.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, practice the present invention toits fullest extent.

EXAMPLES Example 1

Inflammatory pain model: The nociception response was measured using thehind paw withdrawal latency test modified after Hargreaves at al., Pain,32(1):77-88 (1988). Male Sprague-Dowley rats weighing 240-260 g, areindividually housed at UC Davis Animal Resource Facility under standardconditions with free access to food and water, and maintained for atleast 1 week before the experiments. On the day of the experiments, theanimals' basal response is measured and then compounds are topicallyadministered preceding an injection with 10 ug of endotoxin(1Lipopolysaccharide, “LPS”, Sigma-Aldrich, St. Louis, Mo.). Nociceptiveresponse is then measured at 120 minutes post-LPS injection. Compoundsare formulated by dissolving them in ethanol and mixing with cream in aratio of 1:8. Eight animals per group are used. A dose response curve isobtained by administering increasing concentrations of EETs andmeasuring nociceptive response.

Example 2

To determine the cellular receptors for EETs, in vitro bioassays wereconducted using human receptors. From 150 available human receptors, 47were selected on the basis of behavioral observations of animals whenEETs were administered to them. These 47 selected human molecularreceptors were screened using radio-ligand competition assays toidentify potential receptors for EETs. The biological outcome ofimpacting these receptors was compared with the observed behavior toinclude each receptor into the screen. Based on the results of initialscreening, efforts were focused on individual receptors, which werescreened with each of the four regio-isomers of EETs. Several receptorsand EET isomers were ruled out as not exhibiting activity.

Receptor Binding Experiments.

Standard radio-ligand binding competition experiments were conducted on47 human receptors using a final concentration of 10 μM. Percentinhibition of a known potent agonist was reported. The threshold fordefining a “positive” hit was set as 25% inhibition of binding.Receptors which were inhibited by more than 25% in the first screen werefurther investigated by conducting the binding experiments usingindividual isomers of EETs.

Example 3

Bioassays. When animals are treated with lipopolysaccharide (LPS), theyshow a drastic reduction in their withdrawal latencies in pain responseassays. The pain response, however, was restored towards the baselinelevels with the application of increasing concentrations of EETs (doses50,200 and 300 mg/kg).

Analgesic effect of EETs. LPS treatment produces hyperalgesia byreducing the baseline withdrawal latency by two fold. Animals topicallyadministered EETs display significantly less hyperalgesia and theirnociceptive response remains at the baseline level.

Receptor Binding Assays

As noted in the preceding Examples, first round screens were conductedusing 47 human receptors. The receptors were expressed in recombinantmammalian cells. Only the receptors that were significantly inhibited by10 pM of EETs are reported. Related receptor subtypes, however, are alsoincluded in Table 2 to emphasize specificity of EETs to the labeledreceptors. In a second round of screens, the receptors that gavepositive hits in the first round were selected and screened against eachof the four regioisomers of EETs. This experiment was done with 3 μM ofEETs to increase the stringency of the screen. The results aresummarized in Table 3. The activity observed on Dopamine D3 receptor waslost in the second round. However significant inhibition of peripheralbenzodiazepine receptors was observed with three of the four isomers ofEETs (Table 2). Additionally 5,6-EET remained to have the same level ofactivity on Cannabinoid CB2 and Neurokinin NK2 receptors. Tables 2 and 3present the early data we developed, while Table 4 presents morecomplete data.

TABLE 2 Screening of human receptors against a mixture of EETs. %Inhibition IC50 of Control Ref Receptor Specific Binding ReferenceCompound (M) BZD (peripheral) 78 PK11195 2.70E−09 BZD (central) 12diazepam 1.00E−08 Cannabinoid CB1 13 CPS6940 1.00E−09 Cannabinoid C62 26WIN55212-2 7.60E−09 Dopamine D1 20 SCH23390 4.80E−10 Dopamine D2S 16(+)butaclamol 4.40E−09 Dopamine D3 47 (+)butaelamol 8.90E−09 NeurokininNK1 12 [Sar9,Met(02)11]-SP 2.20E−10 Neurokinin NK2 32 [Nie10]-NKA(4-10)9.30E−09 Neurokinin NK3 1 SB 222200 1.00E−08

TABLE 3 Second round of screening using individual EET isomers onreceptors that were significantly inhibited by a mixture of EETs infirst screen. % Inhibition Test of Control IC50 Com- Specific ReferenceRef Receptor pound Binding Compound (M) BZD (peripheral) 5,6-EET 27PK11195 2.5E−09 BZD (peripheral) 8,9-EET 11 PK11196 2.5E−09 BZD(peripheral) 11,12-EET 28 PK11195 2.5E−09 BZD (peripheral) 14,15-EET 45PK11195 2.5E−09 Cannabinoid CB2 5,6-EET 25 WINS5212-2 2.7E−09Cannabinoid CB2 8,9-EET −2 WING5212-2 2.7E−09 Cannabinoid CB2 11,12-EET3 WINS5212-2 2.7E−09 Cannabinoid CB2 14,15-EET 5 WIN55212-2 2.7E−09Dopamine D3 5,6-EET 18 (+)butaclamol 8.7E−09 Dopamine D3 8,9-EET 9(+)butaclamol 8.7E−09 Dopamine D3 11,12-EET 6 (+)butaclamol 8.7E−09Dopamine D3 14,15-EET 10 (+)butaclamol 8.7E−09 Neurokinin NK2 5,6-EET 25[Nle10]- 9.7E−09 NKA(4.10) Neurokinin NK2 8,9-EET 10 [Nle101- 9.7E−09NKA(4-10) Neurokinin NK₂ 11,12-EET 1 [Nle10]- 9.7E−09 NKA(4-10)Neurokinin NK₂ 14,15-EET 8 [Nle10]- 9.7E−09 NKA(4-10)

TABLE 4 Interaction of EETs with selected cellular receptors PeripheralCentral CB₁ CB₂ NK₁ NK₂ NK₃ benzodiazepine benzodiazepine D₃ EET-memixture (μM) >100  19 >100  14 >100 4.6 >100  30 5,6 EET-me (μM) NT  20NT  36 NT 12 NT >100 8,9 EET-me (μM) NT >100 NT >100 NT >100 NT >10011,12 EET-me (μM) NT >100 NT >100 NT 140 NT >100 14,15 EET-me (μM)NT >100 NT >100 NT 12 NT >100 Binding assays were conducted by CEREPaccording to standardized procedures. A mixture of regioisomers of EETswere initially screened broadly for displacing ability of high affinityligands. In a second round the IC₅₀ of the mixture and the individualisomers were determined. Reference compounds and their affinities (M)for respective receptors from left to right were CP 55940 (1.00E−09),WIN 55212-2 (7.60E−09), [Sar9, Met(O2)11]-SP (2.20E−0),[N1e10]-NKA(4-10) (9.30E−9), SB 222200 (1.00E−8), PK 11195 (2.70E−9),Diazepam (1.0E−08), (+) butaclamol (8.90E−09). NT: not tested.

Example 4 Receptor Binding Assays

Receptor binding experiments were contracted to CEREP (Redmond, Wash.).Compounds with encrypted identities were mailed to CEREP. Standardradio-ligand binding competition experiments were conducted initially on47 receptors using a final concentration of 10 μM. Percent inhibition ofa known potent agonist was reported. The threshold for a positive hitwas set as 25% inhibition of binding by CEREP. Receptors which wereinhibited at more than 25% in the first screen were further investigatedby conducting the binding experiments using individual isomers of EETs.

(i) PBR Binding Assays.

For the peripheral benzodiazepine assay, the procedure of Le Fur et al.(Life Sci. 33: 449-457 (1983)) was followed. Briefly, maleSprague-Dawley rats (200 g, Charles River Laboratories, Inc.,Wilmington, Mass.) were sacrificed and hearts were excised. Theventricular tissue was homogenized (1:4 w/v) in cold sucrose (0.25 M),Tris HCl (5 mM, MgCl₂ (1 mM) buffer at pH 7.4. The homogenates were thenfiltered through a double layer of cheese cloth and centrifuged at1,000×g for 10 minutes. The supernatant was recentrifuged at 40,000×gfor 30 minutes. The resulting pellet was resuspended in the incubationbuffer. The binding assays were performed in 50 mM Tris HCl, MgCl₂ 10 mMbuffer pH 7.5 in a final volume of 1 ml containing 0.2 mg of cardiacmembrane protein and the radioactive ligand, [³H] PK 11195(1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinolinecarboxamide, a powerful PBR ligand) and increasing concentrations ofEETs. After 15 minutes at 25° C., the membranes were filtered over GF/Cfilters (Whatman Inc., Florham Park, N.J.) followed by 3×5 ml washeswith cold buffer. Specific binding (95% of total binding for bothligands) was defined as the amount of radioactivity displaced by 1 μMunlabelled RO5-4864 (4′-chlorodiazepam), a ligand known to bind PBR. Theradioactivity in the filters was measured with a scintillation counter.Equilibrium thermodynamic parameters of binding were determinedutilizing classical thermodynamic equations.

(ii) CB2 Binding Assays

For CB2 binding assays, recombinant human receptor protein was expressedin Chinese Hamster Ovary (CHO) cells. The binding of the syntheticcannabinoid receptor agonist [³H] WIN 55212-2((4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one)to cell membranes was determined by incubation of the ligand (0.8 nM)for 2 hours at 37° C. with cells in the cell culture buffer according toMunro et al. (Nature, 365:61-65 (1993)). EETs were added in increasingconcentrations in parallel. The membranes were then filtered over GF/Cfilters (Whatman), followed by 3×5 ml washes with cold buffer. Specificbinding (95% of total binding for both ligands) was defined as theamount of radioactivity displaced by 5 μM of unlabelled WIN 55212-2.

Example 5 Behavioral Nociceptive Testing

Behavioral nociceptive testing was conducted by assessing thermalhindpaw withdrawal latencies (“TWL”) using a commercial Hargreaves(Hargreaves et al., A new and sensitive method for measuring thermalnociception in cutaneous hyperalgesia. Pain 32:77-88 (1988)) apparatus(IITC Life Science Inc., Woodland Hills, Calif.). Male Sprague-Dawleyrats weighing 240-250 g, were individually housed at the UC Davis AnimalResource Facility under standard conditions with free access to food andwater, and maintained for at least 1 week before the experiments. On theday of the experiment, the rats were transferred to a quiet room,acclimated for 1 h, and their baseline responses measured. In pilotexperiments, the intensity of the thermal stimulus was set to produce abaseline TWL of 7-8 s. Following baseline measurements, rats were firsttreated with 200 μl of vehicle or compound-formulated cream by topicalapplication to one hind paw. Compounds (including sEHI, steroidsynthesis inhibitors, steroid receptor antagonists, and cannabinoidreceptor antagonists, as shown in the Figure legends) were formulated bydissolving them in ethanol and mixing with Vanicream® (PharmaceuticalSpecialties, Inc., Rochester, Minn.) in a ratio of 1:9. The cream wasthoroughly massaged across the entire hind paw surface over a 2 minperiod. After 1.5 hours rats were treated with 200 μl of sEHI formulatedcream. Within 10 min of sEHI application, lipopolysaccharide (“LPS”, 10μg in 50 μl 0.9% NaCl) was injected subcutaneously into the plantarsurface of the treated paw. Immediately following LPS injection, animalswere placed in acrylic chambers on a glass platform maintained at atemperature of 30±1° C. for TWL-measurement. During TWL measurement, abeam of radiant heat was focused onto the mid-portion of the plantarsurface of the treated hind paw until the rat moved its stimulatedhindpaw abruptly away from the heat stimulus. The duration of heatapplication necessary to elicit a withdrawal was designated as TWL. Amaximum stimulus duration of 22 s was imposed to prevent tissue damage.Five TWL measurements were taken at 3-4 min interstimulus intervals foreach of the time points following treatment. The three median TWLs wereaveraged for each animal at each time point.

Example 6

Fatty acids and lipid signaling: Lipid molecules are ubiquitousmessengers that are known to participate in intracellular signaling,cell to cell communication and serve as neurotransmitters. Lipidmessengers also regulate specific physiologic functions, one of which isthe transmission of noxious sensory information (pain) in the peripheryand the central nervous system. A significant aspect of the role oflipids in neuronal function is their ability to modify the functionalresponses of ion channels, synaptic transmission and cellular signalingcascades through which neuronal cell function is modified to meetphysiologic demand (Sang, N. Neuroscientist 12:425-434 (2006); Chen, C.et al., Prostaglandins & Other Lipid Mediators 77:65-76 (2005)).Analysis of alterations in the type, amount and organization of lipidscan provide critical information leading to the understanding ofmechanism of action of each molecule, the early diagnosis of disease,identification of the mechanisms underlying the disease process itselfand also can potentially provide an indication of efficacy of specifictreatment regimes. For example it has recently become clear that,despite previous thinking, the kinetic characteristics of ion channelsare intimately related to their dynamic interactions with theirsurrounding lipids and electric field-induced changes in protein-lipidinteractions (De Petrocellis, L. et al., Life Sciences 77:1651-1666(2005)). Thus, the lipid environment is now recognized as a directmodifier of the functional outcome of a transmitted electrical signal.

The arachidonic acid (AA) cascade is a relatively well exploitedbiological path with many therapeutic opportunities, only a limitednumber of which are taken advantage of. Moreover, there is evidence ofthe existence of parallel homologous cascades of other fatty acids,particularly a linoleic acid (LA) cascade. Although direct action of AAon various ionic currents has been demonstrated (Ordway, R. W. et al.,Science 244:1176-1179 (1989)) the released AA is quickly converted todownstream metabolites by prostaglandin synthases, lipoxygenases andcytochrome P450 enzymes in a tissue- and context-dependent manner(Roman, R. Metabolites of Arachidonic Acid in the Control ofCardiovascular Function Physiological Reviews 82:131-185 (2002);Capdevila, J. et al., FASEB Journal 6:731-736 (1992); McGiff, J. C.Annual Review of Pharmacology and Toxicology 31:339-369 (1991)).Eicosanoids, the arachidonic acid-derived lipid mediators, are composedof several classes that include leukotrienes (LT), prostaglandins (PG),thromboxanes (TX), and hydroxy, epoxy and oxo-fatty acids. Theseeicosanoids are formed by various cells and most are thought to actlocally (McGiff, J. C. Annual Review of Pharmacology and Toxicology31:339-369 (1991); Imig, J. Clinical Science (London) 111:21-34 (2006)).Their biological roles include control of vascular tone, plateletaggregation, renal function, hypersensitivity and inflammation; thus,they are of great physiological importance. LA mono-epoxides (EpOMEs)and diols (DiHOMEs) also have many biological activities. For example,they induce vasodilatation and thus appear to regulate blood pressure(Ishizaki, T. et al., Am J Physiol 268:123-128 (1995)), may protectorganisms from infectious diseases (Hayakawa, M. et al., Biochem BiophysRes Commun 137:424-430 (1986)), and may have a role in multiple-organfailure associated with severe burns, acute trauma, and respiratorydistress syndrome (Hayakawa, M. et al., Biochem Int 21:573-579 (1990);Ozawa, T. et al., Am Rev Respir Dis 137:535-540 (1988); Kosaka, K. etal., Mol Cell Biochem 139:141-148 (1994)). EpOMEs may be endogenouschemical mediators regulating vascular permeability (Hennig, B. et al.,Metabolism 49:1006-1013 (2000)).

Epoxy fatty acids and sEH: One of the metabolic fates of AA is theoxidation to EETs by cytochrome P450 epoxygenases. A multitude ofinteresting biological activities are found to be associated with theEETs using in vitro systems (Campbell, W. et al., Circulation Research78:415-423 (1996)). Although EETs other than the 5,6-isomer are quitestable chemically, they are quickly degraded enzymatically with the sEHaccounting in many cases for much of the metabolism. This rapiddegradation has so far made it difficult to associate biological effectswith the administration of EETs and other lipid epoxides particularly invivo. Soluble epoxide hydrolase (sEH, EC 3.3.2.3), a α/β fold hydrolyticenzyme that primarily hydrolyzes epoxides on acyclic systems, is themajor enzyme that biodegrades EETs. By inhibiting sEH to increase theresidence time of EETs, recently it has become clear that major roles ofthe EETs include but are not limited to modulation of blood pressure andmodulation of inflammatory cascades (Spector, A. et al., Progress inLipid Research 43:55-90 (2004); Node, K. et al., Science 285:1276-1279(1999)). There are a number of other biological effects associated withthe EETs, including neurohormone release, modulation of ion channelactivity, cell proliferation, G-protein signaling and a variety ofeffects associated with modulation of NFκB (Spector, A. et al., Progressin Lipid Research 43:55-90 (2004); Node, K. et al., Science285:1276-1279 (1999); Fleming, I. Hypertension 47:629-633 (2006);Feletou, M. et al., Arteriosclerosis, Thrombosis, and Vascular Biology26:1215-1225 (2006)).

We have demonstrated a role of the EETs as modulated by sEH inhibitors(sEHIs) in reducing inflammatory pain (Inceoglu, B. et al., LifeSciences 79:2311-2319 (2006)). The array of biological effects observedwith sEH inhibition illustrates the power of modulating the degradationof chemical mediators. Many of these biological effects can be modulatedby sEHIs but presumably also by the natural eicosanoids and theirmimics, all of which offer therapeutic potential. EETs and possiblyother epoxy fatty acids are clearly regulatory molecules. By way ofmetabolic profiling of oxylipids and prostanoids, work in our laboratoryhas shown that blocking the COX and sEH branches simultaneously resultsin a synergistic decrease in prostaglandin production, and thusinflammation and pain, when a lipopolysaccharide (LPS)-elicited acuteinflammatory model is used (Schmelzer, K. et al., Proc Natl Acad Sci USA103:13646-13651 (2006) (“Schmelzer PNAS 2006”); Schmelzer, K. et al.,Proc Natl Acad Sci USA 102:9772-9777 (2005)). Inhibition of sEH or COX 2results in clear increases in EET concentrations. Our data implies thatat least some of the effects of COX-2 inhibitors may be through anincrease in EETs (Schmelzer PNAS 2006).

Steroidogenesis, AA, EETs, StAR and the peripheral benzodiazepinereceptor: Steroid hormones are synthesized in steroidogenic cells of theadrenal, ovary, testis, placenta, and brain and are required forreproductive function and homeostasis. Acute steroidogenesis, regulatedby trophic hormone stimulation, occurs on the order of minutes and isinitiated by the mobilization and delivery of the substrate for allsteroid hormone biosynthesis, cholesterol, from the outer to the innermitochondrial membrane where it is metabolized to pregnenolone by thecytochrome P450 cholesterol side chain cleavage enzyme, P450scc (Payne,A. H. et al., Overview of Steroidogenic Enzymes in the Pathway fromCholesterol to Active Steroid Hormones, pp 947-970 (2004)).

The essential role of arachidonic acid (AA) in trophichormone-stimulated steroidogenesis has been demonstrated starting in theearly 1980s (Lin, T. Life Sciences 36:1255-1264 (1985) (“Lin 1985”)).Various authors have suggested COX and LOX metabolites of AA wereinvolved in the process (Lin 1985; Dix, C. J. et al., The BiochemicalJournal 219:529-537 (1984); Mercure, F. et al., General and ComparativeEndocrinology 102:130-140 (1996); Campbell, W. B. et al., Journal OfSteroid Biochemistry 24:865-870 (1986)). Stimulatory effects of the P450branch, the epoxygenase products, on steroidogenesis were also reportedin bovine adrenal cells early on (Nishimura, M. et al., Prostaglandins38:413-430 (1989)). In human granulosa cells, low concentrations of EETswere reported to stimulate estradiol secretion (Van Voorhis, B. J. etal., J Clin Endocrinol Metab 76:1555-1559 (1993)). Recently, D. M.Stocco's group reported the identification of EETs as one of the factorsthat stimulate StAR (steroidogenic acute regulatory protein) expressionand steroidogenesis (Wang, X. et al., The involvement of epoxygenasemetabolites of arachidonic acid in cAMP-stimulated steroidogenesis andsteroidogenic acute regulatory protein gene expression, pp 871-878(2006)). StAR protein is one of the candidate proteins proposed asessential for steroidogenesis, possessing all the necessarycharacteristics of the acute regulator (Clark, B. J. et al.,Characterization of the steroidogenic acute regulatory protein (StAR),pp 28314-28322 (1994)). The acute response to hormonal stimulation hasan absolute requirement for de novo protein synthesis (Davis, W. W. etal., The Inhibitory Site Of Cycloheximide In The Pathway Of SteroidBiosynthesis, pp 5153-5157 (1968); Garren, L. D. et al., Studies on theRole of Protein Synthesis in the Regulation of Corticosterone Productionby Adrenocorticotropic Hormone in vivo, pp 1443-1450 (1965)). Inhibitionof protein synthesis blocks hormone-induced steroid synthesis byblocking the delivery of cholesterol to the inner mitochondrial membrane(Farkash, Y. et al., Endocrinology 118:1353-1365 (1986)). Sinceactivation of StAR protein expression is rapid and temporally related tosteroid synthesis, the mRNA and protein quantities of StAR are goodindicators of steroidogenesis.

Although a large body of literature exists on the actions of AA and itsmetabolites on steroid synthesis in the steroidogenic tissues, acutesteroidogenesis in the nervous system is much less known but thought toproceed in parallel to that in steroidogenic cells (Furukawa, A. et al.,Steroidogenic Acute Regulatory Protein (StAR) Transcripts ConstitutivelyExpressed in the Adult Rat Central Nervous System: Colocalization ofStAR, Cytochrome P-450SCC (GYP XIA1), and 3beta-HydroxysteroidDehydrogenase in the Rat Brain, pp 2231-2238 (1998)). In this regard, wehave evidence that inhibition of sEH impacts steroidogenesis, presumablyin the nervous tissues and that sEHI elicited analgesia is through acutemodulation of steroidogenesis.

Another steroidogenesis regulating protein is the peripheralbenzodiazepine receptor (PBR). As the biological roles of the PBR areemerging, the regulation of steroidogenesis among these roles(regulation of cellular proliferation, apoptosis, immunomodulation,porphyrin transport and heme biosynthesis) seems to predominate (Gavish,M. et al., Receptor Pharmacological Reviews 51:629-650 (1999);Papadopoulos, V. L. et al., Neuroscience 138:749-756 (2006)). Ligandbinding to PBR results in the stimulation of mitochondrial pregnenoloneformation (Mukhin, A. G. et al., Mitochondrial Benzodiazepine ReceptorsRegulate Steroid Biosynthesis, pp 9813-9816 (1989)). In addition, potentPBR ligands block inflammation profoundly in several distinct animalmodels of chronic inflammation (Torres, S. R. et al., European Journalof Pharmacology 408:199-211 (2000); Bressana, E. et al., Life Sciences72:2591-2601 (2003)). Several inhibitors of steroid synthesizing enzymescan block the effects of PBR ligands (da Silva, M. et al., Mediators ofInflammation 13:93-103 (2004); Farges, R. et al., Life Sciences74:1387-1395 (2004)).

Indeed Farges et al. showed that the P450scc inhibitor aminoglutethimideblocks the anti-inflammatory effects of PBR ligands in vivo (Farges, R.et al., Life Sciences 74:1387-1395 (2004)). The current proposed mode ofaction for this activity is that PBR complex, which includes StARprotein, VDAC (voltage dependent anion channel 1), PAP7, PKARIα(cAMP-dependent protein kinase) and DBI (diazepam binding inhibitor)proteins, regulates steroid biosynthesis by facilitating the import ofcholesterol from the outer to the inner mitochondrial membrane and thatits acute modulation increases steroid and/or neurosteroid synthesis(Liu, J. et al., Protein-Protein Interactions Mediate MitochondrialCholesterol Transport and Steroid Biosynthesis, pp 38879-38893 (2006)).The import of cholesterol has long been recognized as the first and therate limiting step in steroidogenesis (Papadopoulos, V. L. et al.,Neuroscience 138:749-756 (2006); Bose, H. S. et al., Nature 417:87-91(2002)). Both StAR and PBR proteins seem to be indispensable elements ofthe steroidogenic machinery and they function in a coordinated manner totransfer cholesterol into mitochondria (Hauet, T. et al.,Peripheral-Type Benzodiazepine Receptor-Mediated Action of SteroidogenicAcute Regulatory Protein on Cholesterol Entry into Leydig CellMitochondria, pp 540-554 (2005); Stocco, D. M. et al., MultipleSignaling Pathways Regulating Steroidogenesis and Steroidogenic AcuteRegulatory Protein Expression: More Complicated than We Thought, pp2647-2659 (2005)). In addition, Hauet et al. proposed that PBRactivation is required for StAR expression.

Cholesterol upon entering mitochondria is potentially directed to thesynthesis of specific steroids in each tissue, which is dictated by thepresence and activity of steroid synthesizing enzymes in a particulartissue. In fact, the differential expression and distribution of theseenzymes is proposed to control the non-acute endogenous steroid tone. Achange in the endogenous steroid tone through acute steroidogenesis mayresult with several favorable physiological outcomes includinganxiolysis and analgesia, primarily through the actions of neurosteroidson GABAA conductance in the nervous tissue (Verleye, M. et al.,Pharmacology Biochemistry and Behavior 82:712-720 (2005); Sanna, E. etal., The Journal of Neuroscience 24:6521-6530 (2004)). Neurosteroids3α,5α-THPROG and 3α,5α-THDOC, for example are known to bind to andmodulate GABAA channels which are inhibitory in nature and displayanxiolytic, analgesic, anticonvulsant, sedative, hypnotic andanaesthetic properties (Belelli, D. et al., Nature Reviews Neuroscience6:565-575 (2005)).

One of the most intriguing findings in respect to the role of EETs ininflammation was that EETs, through inhibiting NFκB, areanti-inflammatory (Node, K. et al., Science 285:1276-1279 (1999)). Thisalso holds true in vivo in rats although EETs are algesic in the absenceof inflammatory pain (Inceoglu, B. et al., Life Sciences 79:2311-2319(2006)). As noted elsewhere herein, we found that EETs can displace highaffinity radioligands from a number of cellular receptors previously notknown to be associated with EETs, two of which are the mitochondrial orperipheral benzodiazepine receptor (PBR) and the CB2 receptor. Epoxyfatty acids and their increase in concentration or half life by way ofsEH inhibition causes a favorable shift in the endogenous steroid tonethrough modulation of PBR and StAR, ultimately impacting GABAA channelsand this is at least one of the mechanisms responsible for the observedpowerful anti-inflammatory and/or analgesic effect of EETs and sEHinhibitors.

Pain, and neuropathy: Upon nerve damage the pro-inflammatory cytokines,specifically TNF-α is up regulated in surrounding tissues of nervesincluding the Schwann cells, mast cells and resident macrophages (Myers,R. R. et al., Drug Discovery Today 11:8-20 (2006)). This increase leadsto a pathological process of progressive nerve degeneration which wasrecognized as early as 1850. Wallerian degeneration is highly correlatedwith the development of neuropathic pain (Stoll, G. et al., Journal ofthe Peripheral Nervous System 7:13-27 (2002)). Following nerve injury,nonresident macrophages in response to secreted chemotactic signalsinvade the injury site (Sommer, C. et al., Neuroscience Letters270:25-28 (1999); Zelenka, M. et al., Pain 116:257-263 (2005)). Thisinvasion and the process of nerve degeneration are temporally related tothe peak periods of hyperalgesia in neuropathic pain (Shubayev, V. I. etal., A spatial and temporal co-localization study in painful neuropathy,pp 28-36 (2002)). Further migration of immune cells through theendothelium follows chemotactic signals that are released by injurednerves (Wagner, R. et al., Neuroscience 73:625-629 (1996)). Activatedmacrophages secrete components of the complement cascade, coagulationfactors, proteases, hydrolases, interferons, TNF-α and other cytokines(Zelenka, M. et al., Pain 116:257-263 (2005)). Local TNF-α causesspontaneous electrophysiological activity in surviving nociceptive nervefibers contributing to pain (Wagner, R. et al., Neuroreport 7:2897-2901(1996)). Interestingly, the potential role of arachidonic acidmetabolites have not systematically been investigated in thepathophysiology of neuropathic pain despite the fact that COX-2inhibitors are quite effective in animal models of nerve injury(Bingham, S. et al., Journal of Pharmacology and ExperimentalTherapeutics 312:1161-1169 (2005); Ma, W. et al., Brain Research937:94-99 (2002)). Moreover most animals studies that tested COX-2inhibitors on neuropathic pain when the drug was given before or shortlyfollowing nerve injury showed encouraging results (Bingham, S. et al.,Journal of Pharmacology and Experimental Therapeutics 312:1161-1169(2005); De Vry, J. et al., European J Pharmacology 491:137-148 (2004)).Our limited mechanistic understanding of the pathways involved inneuropathic pain is clearly paralleled in the treatment of thiscondition. Currently, no single treatment options without significantside effects exist for neuropathic pain. A combination ofpharmacological agents that block or attenuate the propagation ofinflammation and potent analgesics are usually prescribed albeit withvariable success (Gilron, I. et al., Canadian Med Assn J 175:265-275(2006)).

The arachidonate cascade is the target for a significant fraction of thepharmaceuticals on the market and includes such NSAID drugs as salicylicacid, ibuprofen, naproxen, and celecoxib. We have found that sEHIs aremore potent at reducing inflammatory eicosanoids in plasma than any ofthe above drugs (Schmelzer PNAS 2006). More importantly, NSAIDs shiftarachidonic acid from one inflammatory cascade to another. In contrast,sEHI shift the blood eicosanoid profile from one propagating andexpanding a pain response to one resolving pain toward a healthy state.

sEHIs and EETs are antinociceptive and analgesic in inflammatory painmodels: Inhibition of sEH has been shown to result in a multitude ofbeneficial effects. One of these intriguing effects is that sEHIsprotect mice from LPS elicited acute inflammation (Schmelzer, K. et al.,Proc Natl Acad Sci USA 102:9772-9777 (2005)). LPS induced mortality,systemic hypotension, and histologically evaluated tissue injuries weresubstantially diminished by administration of urea-based, small-moleculeinhibitors of sEH to mice. Moreover, sEH inhibitors decreased plasmalevels of proinflammatory cytokines and nitric oxide metabolites whilepromoting the formation of lipoxins, thus supporting inflammatoryresolution. These data suggest that sEHIs have therapeutic efficacy inthe treatment and management of acute inflammatory diseases. The sEHIdependant reduction of prostanoid production in this model also suggeststhat inhibition of sEH may attenuate inflammatory pain. This isconfirmed by using two distinct inflammatory pain models where we showedthat inhibitors of sEH are antihyperalgesic.

Hyperalgesia in the LPS-elicited pain model was induced by intraplantarLPS injection and sEH inhibitors were delivered topically. We found thaturea based sEHIs can successfully be delivered through the transdermalroute. The maximal biological effect of sEHI AEPU also corresponds tothe maximum plasma concentration and that sEH inhibitors effectivelyattenuate thermal hyperalgesia and mechanical allodynia in rats treatedwith LPS. In addition, we show that epoxydized arachidonic acidmetabolites, EETs, are also effective in attenuating thermalhyperalgesia in this model. In parallel with the observed biologicalactivity, metabolic analysis of oxylipids showed that inhibition of sEHresulted in a decrease in PGD2 levels and sEH generated degradationproducts of linoleic and arachidonic acid metabolites with a concomitantincrease in epoxides of linoleic acid.

Using a second distinct inflammatory model, hyperalgesia was induced byintraplantar injection of 2% carrageenan (CAR) and sEH inhibitors wereagain delivered topically 20 hours post CAR injection. The sEHI AUDA-beblocked CAR induced local thermal hyperalgesia effectively. AUDA-be notonly had a prophylactic effect in the LPS model, but was also effectivetherapeutically in reversing thermal hyperalgesia. These data show thatinhibition of sEH may become a viable therapeutic strategy to attainanalgesia.

EETs Act on PBR.

Although inhibition of sEH will decrease pain the mechanism of action ofthis effect is largely unknown. A current hypothesis is that inhibitionof sEH leads to increased stability, hence residence time of naturalEETs and that EETs are responsible for the observed biological activity.Therefore we subjected a mixture of regioisomers of EETs to a standardreceptor screening using high affinity radioligands. This assay wasconducted by a contract research organization (CEREP). We selected asubset of 48 cellular receptors based on the behaviors of the sEHknockout mice and sEHI treated rats. Four of these receptors wereinhibited by EETs with micromolar affinities.

sEHI Elicited Analgesia is Blocked by Inhibition of Steroid/NeurosteroidSynthesis

Based on the functions of PBR, we hypothesized that the interaction ofEETs with this receptor may cause an increase in steroid production inthe periphery and neurosteroid production in the brain. We usedpharmacological inhibitors of steroid synthesis to test this hypothesis.Specifically, aminogluthetimide (AGL), effectively blocks all steroidsynthesis by inhibiting the first enzyme, P450scc, in the steroidsynthesis pathway. When topically administered to rats, AGL completelyblocked the antihyperalgesic action of sEHI AEPU in the LPS-elicitedinflammatory pain model. We selected AEPU for these tests because AEPUis less likely to be an EET mimic than are some other sEHI, inparticular AUDA-be, because of its structural properties (i.e. thepolyglycol secondary pharmacophore). Additionally, another inhibitor,finasteride, blocks 5α reductase and stops the steroid biosynthesis byblocking the conversion of testosterone to dihydrotestosterone in thecase of steroids and the conversion of progesterone to allopregnanolonein the case of neurosteroids. Finasteride also blocked theanti-hyperalgesic activity of AEPU. In contrast, however, anon-steroidal estrogen receptor antagonist, tamoxifen, a dualprogesterone/glucocorticoid receptor antagonist, mifepristone, anandrogen receptor antagonist, nilutamide, and an aldosterone receptorantagonist, spironolactone, did not have any impact on theantihyperalgesic action of AEPU, indicating that sEHIs and/or EETs arenot acting through these steroid receptors.

As shown in FIG. 3, oxylipin analysis from animals in these tests showedthat the PGE2 levels did not correlate well with the LPS elicitedthermal hyperalgesia bioassay in animals treated with steroid synthesisinhibitor+sEHI. For example PGE2 levels were significantly lower inanimals that received AGL+LPS+AEPU than in animals treated with AEPU+LPSor LPS only, whereas these animals were clearly hyperalgesic despite theadministration of AEPU. See, FIG. 3. This means that inhibition of sEHis not only effective against inflammatory pain but also effective inother types of pain that are not necessarily modulated by prostanoidlevels. Indeed, non-inflammatory types of pain are known to be not welladdressed by COX inhibitors. The levels of EETs and DHETs, however,correlated well with the nociceptive end result. AGL significantlyreduced levels of EETs and increased levels of DHETs inAGL+AEPU-administered animals compared to AEPU-administered animals,indicating that EETs are responsible for the anti-hyperalgesic activity.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of relieving a condition selected from the group consistingof anxiety, panic attacks, agitation, status epilepticus, other forms ofepilepsy, symptoms of alcohol or opiate withdrawal, insomnia, or maniain a subject in need thereof, said method comprising administering tosaid subject an effective amount of an agent or agents selected from thegroup consisting of a cis-epoxyeicosantrienoic acid (“EET”), aninhibitor of soluble epoxide hydrolase (“sEH”), and a combination of anEET and an inhibitor of sEH, thereby relieving said condition in saidsubject. 2-3. (canceled)
 4. A method of claim 1, wherein the agent is aninhibitor of sEH.
 5. A method of claim 1, wherein said condition isanxiety.
 6. A method of inhibiting growth of cancer cells expressingperipheral benzodiazepine receptors (PBR) or CB2 receptors, said methodcomprising contacting said cells with an effective amount of an agent oragents selected from the group consisting of a cis-epoxyeicosantrienoicacid (“EET”), an inhibitor of soluble epoxide hydrolase (“sEH”), and acombination of an EET and an inhibitor of sEH, thereby inhibiting thegrowth of said cancer cells.
 7. A method of claim 6, wherein the cancercells are glioma cells.
 8. A method of claim 6, wherein the cells areastrocytoma cells.
 9. A method of claim 6, wherein the cells are breastcancer cells.
 10. A method of claim 6, wherein the agent is an EET. 11.A method of claim 6, wherein the EET is selected from the groupconsisting of 14,15-EET, and 11,12-EET.
 12. A method of claim 6, whereinthe agent is an inhibitor of sEH.
 13. A method of claim 6, wherein EETor said inhibitor of sEH, or both, are contained in a material whichreleases said EET or said inhibitor, or both, over time.
 14. A method ofreducing oxygen radical damage to cells, said method comprisingcontacting said cells with an effective amount of an agent or agentsselected from the group consisting of a cis-epoxyeicosantrienoic acid(“EET”), an inhibitor of soluble epoxide hydrolase (“sEH”), and acombination of an EET and an inhibitor of sEH, thereby reducing oxygenradical damage to said cells.
 15. A method of claim 14, wherein theagent is an EET.
 16. A method of claim 14, wherein the EET is selectedfrom the group consisting of 14,15-EET, 8,9-EET and 11,12-EET.
 17. Amethod of claim 14, wherein the agent is an inhibitor of sEH.
 18. Amethod of claim 14, wherein EET or said inhibitor of sEH, or both, areadministered by applying to the skin a topical formulation comprisingsaid EET or said inhibitor of sEH or both.
 19. A method of claim 18,wherein said topical formulation further comprises a sunscreen orsunblock.
 20. A method of relieving irritable bowel syndrome (IBS) in asubject in need thereof, said method comprising administering to saidsubject an effective amount of an agent or agents selected from thegroup consisting of a cis-epoxyeicosantrienoic acid (“EET”), aninhibitor of soluble epoxide hydrolase (“sEH”), and a combination of anEET and an inhibitor of sEH, thereby relieving IBS in said subject.21-22. (canceled)
 23. A method of claim 20, wherein the agent is aninhibitor of sEH.